De Novo Design of Potent and Selective Interleukin Mimetics

ABSTRACT

De novo designed polypeptides that bind to IL-2 receptor β   c  heterodimer (IL-2Rβ   c ), IL-4 receptor α   c  heterodimer (IL-4Rα   c ), or IL-13 receptor α subunit (IL-13Rα) are disclosed, as are methods for using and designing the polypeptides.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSerial Nos. 62/689,769 filed Jun. 25, 2018 and 62/768,733 filed Nov. 16,2018, each incorporated by reference herein in its entirety.

BACKGROUND

The considerable potential of central immune cytokine interleukins suchas IL-2 and IL-4 for cancer treatment has sparked numerous efforts toimprove their therapeutic properties by mutation and/or chemicalmodification. However, because these approaches are closely tied tonative IL-2 or IL-4, they cannot eliminate undesirable properties suchas low stability and binding to the IL-2 receptor α subunit (IL-2Rα), toIL-4 receptor α

_(c) heterodimer (IL-4Rα

_(c)), or to IL-13 receptor α subunit (IL-13Rα).

SUMMARY

In one aspect, a method is provided. A computing device determines astructure for a plurality of residues of a protein where the structureof the plurality of residues provides a particular receptor bindinginterface. The computing device determines a plurality of designedresidues using a mimetic design protocol provided by the computingdevice, wherein the plurality of designed residues provide theparticular receptor binding interface, and wherein the plurality ofdesigned residues differ from the plurality of residues.

The computing device determines one or more connecting helix structuresthat connect the plurality of designed residues. The computing devicedetermines a first protein backbone for the protein by assembling theone or more connecting helix structures and the plurality of designedresidues over a plurality of combinations. The computing device designsa second protein backbone for the protein for flexibility and low energystructures based on the first protein backbone. The computing devicegenerates an output related to at least the second protein backbone.

Also included are non-naturally occurring proteins prepared by themethods described herein. The non-naturally occurring proteins can becytokines, for example, non-naturally occurring IL-2 or IL-4 (alsoreferred to herein as IL-2, IL-2/15 mimetics or IL-4 mimetics).

In another aspect, a computing device is provided. The computing deviceincludes one or more processors; and data storage that is configured tostore at least computer-readable instructions that, when executed by theone or more processors, cause the computing device to perform functions.The functions include: determining a structure for a plurality ofresidues of a protein that provides a particular receptor bindinginterface; determining a plurality of designed residues using a mimeticdesign protocol, wherein the plurality of designed residues provide theparticular receptor binding interface, and wherein the plurality ofdesigned residues differ from the plurality of residues; determining oneor more connecting helix structures that connect the plurality ofdesigned residues; determining a first protein backbone for the proteinby assembling the one or more connecting helix structures and theplurality of designed residues over a plurality of combinations;designing a second protein backbone for the protein for flexibility andlow energy structures based on the first protein backbone; andgenerating an output related to at least the second protein backbone forthe protein.

In another aspect, a non-transitory computer-readable medium isprovided. The non-transitory computer-readable medium is configured tostore at least computer-readable instructions that, when executed by oneor more processors of a computing device, cause the computing device toperform functions. The functions include: determining a structure for aplurality of residues of a protein that provides a particular receptorbinding interface; determining a plurality of designed residues using amimetic design protocol, wherein the plurality of designed residuesprovide the particular receptor binding interface, and wherein theplurality of designed residues differ from the plurality of residues;determining one or more connecting helix structures that connect theplurality of designed residues; determining a first protein backbone forthe protein by assembling the one or more connecting helix structuresand the plurality of designed residues over a plurality of combinations;designing a second protein backbone for the protein for flexibility andlow energy structures based on the first protein backbone; andgenerating an output related to at least the second protein backbone forthe protein.

In another aspect, a device is provided. The device includes: means fordetermining a structure for a plurality of residues of a protein thatprovides a particular receptor binding interface; means for determininga plurality of designed residues using a mimetic design protocol,wherein the plurality of designed residues provide the particularreceptor binding interface, and wherein the plurality of designedresidues differ from the plurality of residues; means for determiningone or more connecting helix structures that connect the plurality ofdesigned residues; means for determining a first protein backbone forthe protein by assembling the one or more connecting helix structuresand the plurality of designed residues over a plurality of combinations;means for designing a second protein backbone for the protein forflexibility and low energy structures based on the first proteinbackbone; and means for generating an output related to at least thesecond protein backbone for the protein.

In another aspect, non-naturally occurring polypeptides are providedcomprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence at least 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence at least 25%%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to YAFNFELI (SEQ ID NO:2);

(d) X4 is a peptide comprising the amino acid sequence at least 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to ITILOSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and wherein the polypeptide binds to IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)), IL-4 receptor α

_(c) heterodimer (IL-4Rα

_(c)), or IL-13 receptor α subunit (IL-13Rα).

In other aspects are provided pharmaceutical compositions comprising oneor more polypeptide disclosed herein and a pharmaceutically acceptablecarrier, recombinant nucleic acids encoding a polypeptide disclosedherein, expression vectors comprising the recombinant nucleic acidsdisclosed herein, and recombinant host cells comprising one or moreexpression vector disclosed herein. In a further aspect, methods fortreating cancer are provided, comprising administering to a subjecthaving cancer one or more polypeptide, recombinant nucleic acid,expression vector comprising the recombinant nucleic acid, and/orrecombinant host cells disclosed herein or a pharmaceutical compositionthereof in an amount effective to treat the tumor.

DESCRIPTION OF THE DRAWINGS

The following figures are in accordance with example embodiments:

FIG. 1A-1D. Computational design of de novo cytokine mimetics. FIG. 1A)The designed mimetics have four helices; three mimetic IL-2 interactionswith hIL-2Rβ

_(c), while the fourth holds the first three in place. Top: in the firstgeneration of designs, each of the core elements of IL-2 (helices H1-H4)were independently idealized using fragment-assembly from a clusteredideal fragment database (size: 4 a.a.); bottom: in the second generationof designs the core elements were instead built using parametricequations that recapitulate the shape of each disembodied helix,allowing changes in the length of each helix by +/−8 a.a.; FIG. 1B)Pairs of helices were reconnected using ideal loop fragments (size: 4a.a. or 7 a.a., for gen-1 and gen-2 respectively, see Methods),representative examples are shown with newly built elements connectingeach pair of helices; FIG. 1C) The helix hairpins generated in FIG. 1Bwere assembled in all possible combinations to generate fully connectedprotein backbones; FIG. 1D) The designs and experimentally maturedversions were tested for binding by yeast display, and those exhibitinghigh affinity binding were recombinantly expressed (E. coli) and testedfor binding using surface plasmon resonance and IL-2 like phospho-STAT5(pSTAT5) signaling. The results for 3 designs of the first generationand 10 designs from the second generation are shown in the 2D-plot insolid symbols. The open star is Neoleukin-2/15, the arrow originates inits parent (unoptimized) design.

FIG. 2A-2C. Characterization of neoleukin-2/15. FIG. 2A) From top tobottom: In surface plasmon resonance experiments, neoleukin-2/15 doesnot bind human or murine IL-2Rα, but binds both human and murine IL-2Rβwith similar affinity (K_(d)˜11.2 nM and 16.1 nM, for human and micereceptor, respectively). Like natural IL-2, neoleukin-2/15 binds poorlyto the

_(c) receptor, and exhibits cooperative binding for both human andmurine IL-2Rβ

_(c) (K_(d)˜18.8 nM and 38.4 nM, for the human and mice heterodimericreceptor, while the Kd of native hIL-2 and Super-2 are ˜193.6 nM and300.9 nM, see Table E1). FIG. 2B) top: In-vitro pSTAT5 signaling studiesdemonstrate that neoleukin-2/15 elicits IL-2-like signaling in humancells (EC₅₀), and activates with ˜identical potency (EC₅₀˜73.0 pM and49.2 pM on CD25+ and CD25− cells, respectively) human YT-1 NK cells withor without IL-2Rα expression (CD25); bottom: similarly ex vivoexperiments in murine CD4+ primary cells demonstrate that neoleukin-2/15can also elicit potent IL-2 like signaling in murine cells, and isindependent of IL-2Rα expression (EC₅₀˜24 pM and 129 pM on CD25+ andCD25− cells, respectively); FIG. 2C) top: binding experiments (OCTET)show that neoleukin-2/15 can be incubated for 2 hours at 80° C. withoutany noticeable loss of binding, whereas human and murine IL-2 quicklylose activity; bottom: an ex vivo experiment on cultured murinesplenocytes that require IL-2 for survival, demonstrates thatneoleukin-2/15 incubated at 95° C. for 1 hour still drives cell survivaleffectively (˜70% relative luminescence, at 10 ng/ml), while mIL2 andSuper-2 are virtually inactive (˜10% and 0.1%, respectively at 10ng/ml).

FIG. 3A-3E. Structure of neoleukin-2/15 (Neo-2/15) and its ternarycomplex with mIL-2Rβ

_(c). FIG. 3A) Top: structural alignment of neoleukin-2/15 (Neo-2/15)chain A with the design model (r.m.s.d. 1.11 Å for 100 Cα atoms);bottom: detail of interface helices H1, H3 and H4 (numbered according tohIL-2, see FIG. 1). The interface side chains are shown in sticks; FIG.3B) crystallographic structure of the ternary complex of Neo-2/15 withmIL-2Rβ and γ_(c) (r.m.s.d 1.27 Å for the 93 modeled Cα atoms ofNeo-2/15 in the ternary complex); FIG. 3C) structural alignment ofmonomeric Neo-2/15 (chain A) with Neo-2/15 in the ternary complex(r.m.s.d 1.71 Å for the 93 modeled Cα atoms in the ternary complex).Helix H4 shows an approximately 4.0+shift of helix H4 in theternary-complex structure compared to the monomeric crystal structure;FIG. 3D) crystallographic structure of: hIL-2 (cartoon representation).The regions that interact with the IL-2Rβ and γ_(c) are denoted. Theloop-rich region from hIL-2 that interacts with IL-2Rα does not exist inthe de novo mimetic Neo-2/15. FIG. 3E): crystallographic structure ofneoleukin-2/15 from the ternary complex in “b)” (cartoonrepresentation). The regions that interact with the IL-2Rβ and γ_(c) aredenoted. The loop-rich region from hIL-2 that interacts with IL-2Rα doesnot exist in the de novo mimetic Neo-2/15.

FIG. 4A-4G. Immunogenicity, immunostimulatory and therapeutic activityof neoleukin-2/15. FIG. 4A) Dose escalation effect of neoleukin-2/15(Neo-2/15) in naive mice T cells. Naive C57BL/6 mice were treated dailywith neoleukin-2/15 or mIL-2 at the indicated concentrations (n=2-3 pergroup). After 14 days, spleens were harvested and analyzed by flowcytometry using the indicated markers. The bar plot shows that mIL-2enhanced CD4+ Treg expansion in a dose dependent fashion, while Neo-2/15had little or no effect in expansion of Treg cells. Neoleukin-2/15 drovea higher CD8+:Treg ratio compared to mIL-2; FIG. 4B) Effect of Neo-2/15in mice in an airway inflammation model (20 μg/day/mouse, 7 days).Similar to naive mice, Neo-2/15 does not increase the frequency ofantigen-specific CD4+ Foxp3+ T_(regs) in the lymphoid organs, and iscomparably effective to mIL-2 in increasing the frequency of lungresident (Thy1.2− by intravascular labeling) CD8+ T cells; FIG. 4C)Neoleukin-2/15 does not have detectable immunogenicity. C57BL/6 micewere inoculated with 5×10⁵ B16F10 cells by subcutaneous injection.Starting on day 1, mice were treated daily with neoleukin-2/15 (10 μg)or equimolar mIL-2 by intraperitoneal (i.p.) injection (n=10 for eachgroup). After 14 days, serum (antiserum) was collected and IgG wasdetected by ELISA in plates coated with fetal bovine serum (FBS 10%,negative control), neoleukin-2/15, mIL-2, hIL-2, or Ovalbumin (OVA) asnegative control (the dotted line shows the average of the negativecontrol). Anti-Neo-2/15 polyclonal antibody was used as positive control(black, n=2) and did not cross react with mIL-2 or h-IL2; FIG. 4D)C57BL/6 mice were immunized with 500 μg KO Neo-2/15 in complete Freund'sadjuvant and boosted on days 7 and 15 with 500 μg KO Neo-2/15 inincomplete Freund's adjuvant. Reactivity against KO Neo-2/15 and nativeNeo-2/15, as well as cross-reactivity with mouse IL-2 were determined byincubation of serum (diluted 1:1,000 in PBS) with plate-bound KONeo-2/15, Neo-2/15 or mouse IL-2 as indicated. Serum binding wasdetected using an anti-mouse secondary antibody conjugated to HRPfollowed by incubation with TMB. Data are reported as optical density at450 nm. Top, naive mouse serum; bottom, immunized mouse serum. FIG.4E-4G) Therapeutic efficacy of Neoleukin-2/15: FIG. 4E) BALB/C mice wereinoculated with CT26 tumors. Starting on day 6, mice were treated dailywith i.p. injection of mIL-2 or neoleukin-2/15 (10 μg), or were leftuntreated (n=5 per group). Tumor growth curves (top, show only data forsurviving mice). Survival curves (bottom). Mice were euthanized whenweight loss exceeded 10% of initial weight or when tumor size reached1,300 mm³. FIG. 4F) C57BL/6 mice were inoculated with B16 tumors as in“a)”. Starting on day 1, mice were treated daily with i.p. injection ofneoleukin-2/15 (10 μg) or equimolar mIL-2 (n=10 per group). Twice-weeklytreatment with TA99 was added on day 3. Mice were euthanized when weightloss exceeded 10% of initial weight or when tumor size reached 2,000mm³. Tumor growth curves (top and bottom left). Survival curve, insetshows average weight change (top right). Quantification of cause ofdeath (bottom right). FIG. 4G) Neo-2/15 elicits a higher CD8+:Treg ratiothan mouse IL-2. C57BL/6 mice were inoculated with B16 tumors andtreated by daily i.p. injection as indicated. Treatment with TA99(bottom plot) was started on day 5 and continued twice-weekly. Tumorswere harvested from mice when they reached 2,000 mm³ and analyzed byflow cytometry. The CD8:Treg cell ratio was calculated by dividing thepercentage CD45⁺ CD3⁺ cells that were CD8⁺ by the percentage that wereCD4⁺ CD25⁺ FoxP3⁺.

FIG. 5A-5D. Therapeutic effect of neoleukin-2/15 on colon cancer. FIG.5A) BALB/C mice were inoculated with CT26 tumors. Starting on day-9 andending on day-14, mice were treated daily with i.p. injection of mIL-2or neoleukin-2/15 at the specified concentrations, or were leftuntreated (n=5 per group). Tumor growth curves (top, show only data forsurviving mice). Survival curves (bottom). Mice were euthanized whenweight loss exceeded 10% of initial weight or when tumor size reached1,300 mm³. FIG. 5B-5D) The bar-plots compare the T cell populations forBALB/C mice (n=3 per group) that were inoculated with CT26 tumors andtreated starting from day-6 with by daily i.p. injection of 10 μg ofNeolukin-2/15 or 10 μg mIL-2 or no-treatment (No Tx). On day-14 thepercentage of Treg cells (CD4⁺ CD45⁺ FoxP3⁺, top graph) and CD8:Tregcell ratio ((CD45⁺ CD3⁺ CD8⁺)/Treg, bottom graph) was assessed in: FIG.5B) tumors, FIG. 5C) neighboring inguinal lymph node (LN), and FIG. 5D)spleen.

FIG. 6A-6D. Therapeutic effect of neoleukin-2/15 on melanoma. FIG.6A-6E) Tumor growth curves (bottom) and survival curves (top) forC57BL/6 mice that were inoculated with B16 tumors and treated with low(1 μg/mice/day, a-b) or high doses of neoleukin-2/15 (10 μg/mice/day,c-d). Starting on day 1, mice (n=5 per group) were treated daily withi.p. injection of FIG. 6A): single agent neoleukin-2/15 at 1 μg/mice orequimolar mIL-2 (n=5 per group), or FIG. 6B): the same treatments incombination with a twice-weekly treatment with TA99 (started on day 5).Mice were euthanized when tumor size reached 2,000 mm³. C57BL/6 micewere inoculated with B16 tumors and treated by daily i.p. injection asindicated. FIG. 6C-6D) Similar to “a-b)”, but starting on day 4, micewere treated daily with i.p. injection of 10 μg/mouse of neoleukin-2/15,or equimolar mIL-2, either alone FIG. 6C) or in combination withtwice-weekly TA99 started on day 4 FIG. 6D). Mice were euthanized whentumor size reached 2,000 mm³. The therapeutic effect of Neoleukin-2/15is dose dependent (higher doses are better) and is potentiated in thepresence of the antibody TA99. The experiments were performed once. Inall the growth curves, data are mean±s.e.m. Results were analysed byone-way ANOVA (95% confidence interval), except for survival curves thatwere assessed using the Mantel-Cox test (95% confidence interval).

FIG. 7A-7C. Reengineering of neoleukin-2/15 into a human interleukin-4(hIL-4) mimetic (neoleukin-4). FIG. 7A) Neo-2/15 structurally alignedinto the structure of IL-4 in complex with IL-4Rα and

_(c) (from PDB code 3BPL). Fourteen IL-4 residues that contact IL-4Rαand that were grafted into Neo-2/15 are labeled. FIG. 7B) Neoleukin-4(Neo-4), a new protein with sixteen amino acid mutations compared toNeo-2/15. These mutations are labeled; thirteen of these were derivedfrom the IL-4 residues depicted in panel “a)” that mediate contact withIL-4Rα, and three of them (H8M, K68I and I98F, underlined in the figure)were introduced by directed evolution using random mutagenesis andscreening for high binding affinity variants. FIG. 7C) Biolayerinterferometry data showing that Neo-4, like IL-4, binds to IL-4Rαalone, has no affinity for

_(c) alone, but binds to

_(c) when IL-4Rα is present in solution.

FIG. 8A-8B. Stimulatory effect of Neoleukin-2/15 on human CAR-T cells.FIG. 8A) Anti-CD3/CD28 stimulated or FIG. 8B) unstimulated human primaryCD4 (top) or CD8 (bottom) T cells were cultured in indicatedconcentrations of human IL2 or neoleukin-2/15. T cell proliferation ismeasured as fold change over T cells cultured without IL2 supplement.Neo-2/15 is as effective as native IL-2 at inducing proliferation ofstimulated CAR-T cells, and more effective than native IL-2 at inducingproliferation of unstimulated CAR-T cells, particularly of unstimulatedCD8 CAR-T cells.

FIG. 9A-9D. Overall sequence conservation in binding residues for eachof the four common helices, combining information from three differentde novo-designed IL-2 mimics. Sequence logos were generated usingcombined data from binding experiments (using the heterodimeric mouseIL-2Rβγc) from three independent SSM mutagenesis libraries forG2_neo2_40_1F_seq27, G2_neo2_40_1F_seq29 and G2_neo2_40_1F_seq36 (FIGS.11-13). All of these proteins are functional high-affinity de novomimetics of mouse and human IL-2, some having topologies that differfrom that of Neo-2/15, but all containing the four Helices H1 (FIG. 9A;Neo-2/15 1-22 is SEQ ID NO:248, IL-2 6-27 is SEQ ID NO:249, IL-15 1-15is SEQ ID NO:250), H3 (FIG. 9B; Neo-2/15 34-55 is SEQ ID NO:251, IL-282-103 is SEQ ID NO:252, IL-15 59-80 is SEQ ID NO:253), H2′ (FIG. 9C;Neo-2/15 58-76 is SEQ ID NO:254, IL-2 50-68 is SEQ ID NO:255, IL-1534-52 is SEQ ID NO:256) and H4 (FIG. 9D; Neo-2/15 80-100 is SEQ IDNO:257, IL-2 111-131 is SEQ ID NO:258, IL-15 93-113 is SEQ ID NO:259).The logos show the combined information for each helix independently.Below each logo, a line graph shows the probability score (higher meansmore conserved) for each amino acid in the Neo-2/15 sequence. The solidhorizontal line highlights positions where the Neo-2/15 amino acid has aprobability score ≥30% (that is, these amino acids contribute moregenerally to receptor binding as they are globally enriched in thebinding populations across all of the de novo IL-2 mimics tested). Thetopology of each helix in Neo-2/15 is shown left of each logo. Thesequences of the Neo-2/15 helices and those of the corresponding helices(structurally aligned) in human IL-2 and IL-15 are shown below thegraphs, highlighting the distinctiveness of the Neo-2/15 helices andbinding interfaces.

FIG. 10A-10D. Experimental optimization of G1_neo2_40. FIG. 10A-10C)Heatmaps for G1_neo2_40 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 10A) 50 nM, FIG. 10B) 2 nM, and FIG. 10C) 0.1 nMIL-2Rβ

_(c) heterodimer. Based on these enrichment data, a combinatoriallibrary was designed with nucleotide diversity 1.5×10⁶. FIG. 10D) Aminoacid residues available in the initial combinatorial library aredepicted indicating residues predicted to be advantageous (shown abovethe original sequence) and deleterious (shown below the originalsequence; in the depiction of the original sequence, black indicatesresidues that are represented in the combinatorial library and gray,residues not represented in the combinatorial library.

FIG. 11A-11E. Experimental optimization of G2_neo2_40_1F_seq27. Heatmapsfor G2_neo2_40_1F_seq27 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 11A) 10 nM, FIG. 11B) 1 nM, FIG. 11C) 0.1 nM, andFIG. 11D) 0.1 nM IL-2Rβ

_(c) heterodimer. Based on these enrichment data, a combinatoriallibrary was designed with nucleotide diversity 5.3×10⁶. FIG. 11E) Aminoacid residues available in the initial combinatorial library aredepicted indicating residues predicted to be advantageous; blackindicates residues in the starting sequence represented in thecombinatorial library.

FIG. 12A-12E. Experimental optimization of G2_neo2_40_1F_seq29. Heatmapsfor G2_neo2_40_1F_seq29 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 12A) 10 nM, FIG. 12B) 1 nM, FIG. 12C) 0.1 nM, andFIG. 12D) 0.1 nM IL-2Rβ

_(c) heterodimer. Based on these enrichment data, a combinatoriallibrary was designed with nucleotide diversity 2.9×10⁶. FIG. 12E) Aminoacid residues available in the initial combinatorial library aredepicted indicating residues predicted to be advantageous; blackindicates residues in the starting sequence represented in thecombinatorial library.

FIG. 13A-13E. Experimental optimization of G2_neo2_40_1F_seq36. Heatmapsfor G2_neo2_40_1F_seq36 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 13A) 10 nM, FIG. 13B) 1 nM, FIG. 13C) 0.1 nM, andFIG. 13D) 0.1 nM IL-2Rβ

_(c) heterodimer. Based on these enrichment data, a combinatoriallibrary was designed with nucleotide diversity 2.7×10⁶. FIG. 13E) Aminoacid residues available in the initial combinatorial library aredepicted indicating residues predicted to be advantageous; blackindicates residues in the starting sequence represented in thecombinatorial library.

FIG. 14A-14B. Circular Dichroism (CD) thermal denaturation experimentsfor multiple IL-2/IL-15 de novo designed mimetics, generation-1. FIG.14A) Thermal denaturation curves and FIG. 14B) wavelength scans.

FIG. 15A-15B. Circular Dichroism (CD) thermal denaturation experimentsfor multiple IL-2/IL-15 de novo designed mimetics, generation-1experimentally optimized. FIG. 15A) Thermal denaturation curves and FIG.15B) wavelength scans.

FIG. 16A-16D. Circular dichroism thermal melts for IL-2/IL-15 mimeticdesigns generation-2. FIG. 16A and FIG. 16C) Thermal denaturation curvesand FIG. 16B and FIG. 16D) wavelength scans.

FIG. 17A-17C. Expression, purification, and thermal denaturationcharacterization of neoleukin-2/15. FIG. 17A) SDS Tris-Tricine gelelectrophoresis showing expression and purification over affinitycolumn. FIG. 17B) Circular dichroism at 222 nm during thermal meltingfrom 25° C. to 95° C., showing robust temperature stability. FIG. 17C)Circular dichroism wavelength scans at 25° C., 95° C. and then again 25°C., showing that neoleukin-2/15 does not fully melt at 95° C. andrefolds fully after cooling back to 25° C.

FIG. 18A-18D. Single disulfide stapled variants of neoleukin-2/15 withhigher thermal stability. Structural model of disulfide stabilizedvariants of Neoleukin-2/15 are shown with positions of the mutatedresidues labeled and the disulfide bond shown. Two strategies were usedto generated the disulfide variants: FIG. 18A) internal placement atresidues 38 and 75 and terminal linkage; FIG. 18B) for the terminalvariant, three residues were added to each terminus in order to limitany distortions to the starting structure that would otherwise berequired to form the disulfide bond. CD spectra at 25° C., 95° C. and25° C. after cooling for the internal and terminal disulfide variantsare shown below their structural models. Both variants show very littlesignal loss at 95° C. and complete refolding upon cooling; FIG. 18C)thermal melts of each variant were performed by monitoring CD signal at222.0 nm over a range of temperatures. Each of the disulfide variantsshows improved stability relative the native; FIG. 18D) binding strengthof each variant to IL-2Rβγ_(c) was measured by biolayer interferometry.Contrary to disrupting the binding interaction, these data show theintroduction of the disulfide bond improves the binding of the mimeticsto IL-2Rβγ_(c). Both disulfide-bonded variants exhibit an improvement inbinding IL-2Rβγ_(c) (Kd˜1.3±0.49 and 1.8±0.26 nM, for the internal andexternal disulfide-staples, respectively, compared to 6.9±0.61 nM forNeo-2/15 under the same experimental conditions), which is consistentwith the expected effect of disulfide-induced stabilization of theprotein's binding site.

FIG. 19A-19B. Robustness of neoleukin-2/15 to single-point cysteinemutants on non-binding interface positions. FIG. 19A) Schematic showingpoint mutant positions in neolukin-2/15 that can individually be mutatedto cysteine without interfering with expression of the protein orbinding to IL-2Rβγ_(c). Positions were chosen to avoid interference withreceptor binding. FIG. 19B) Association kinetics of Neolukin-2/15cysteine mutants with IL-2Rβγ_(c) measured using biolayerinterferometry. All of the variants associate with receptorapproximately similarly to Neo-2/15.

FIG. 20A-20C. Expression, purification, and thermal denaturationcharacterization of neoleukin-4. FIG. 20A) SDS Tris-Tricine gelelectrophoresis showing expression and purification over affinitycolumn. FIG. 20B) Circular dichroism at 222 nm during thermal meltingfrom 25° C. to 95° C., showing robust temperature stability. FIG. 20C)Circular dichroism wavelength scans at 25° C., 95° C. and then again 25°C., showing that neoleukin-4 does not fully melt at 95° C. and refoldsfully after cooling back to 25° C.

FIG. 21A-21D. Cytokine levels in non-human primates response to Neo-2/15or Neo-2/15-PEG. Two non-human primates (NHP) per group, one male andone female per group, were assigned to treatment with either vehicle,Neo-2/15 or Neo-2/15-PEG (comprising Neo-2/15 with a single cysteinemutation of E62C conjugated to PEG40K). Animals were administered either0 (vehicle), 0.1, 0.2 or 0.3 mg/kg of Neo-2/15, or 0.05, 0.10 or 0.15mg/kg of Neo-2/15-PEG, by intravenous bolus. Animals treated withNeo-2/15 PEG were administered by intravenous bolus. Cytokine sampleswere taken 0, 4, 8 and 24 hours post dose. Cytokine serum samples wereprepared and frozen at <−70° C. and shipped for analysis where sampleswere analyzed through a Luminex multiplex immunoassays system. Severalcytokines, including IL-10 (FIG. 21A-21B) and IL-15 (FIG. 21C-21D)demonstrated marked differences in the time course of cytokineproduction, consistent with a more sustained pharmacodynamic effect forthe PEGylated molecule.

FIG. 22: A block diagram of an example computing network.

FIG. 23A: A block diagram of an example computing device.

FIG. 23B: A block diagram of an example network of computing devicesarranged as a cloud-based server system.

FIG. 24: A flowchart of a method.

DETAILED DESCRIPTION

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”. Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural or singular number, respectively.Additionally, the words “herein,” “above” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of thisapplication.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine(Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In one aspect, the invention provides non-naturally occurringpolypeptides comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence at least 25%identical to EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence at least 25%identical to YAFNFELI (SEQ ID NO:2);

(d) X4 is a peptide comprising the amino acid sequence at least 25%identical to ITILQSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)) IL-4 receptor α

_(c) heterodimer (IL-4Rα

_(c)), or IL-13 receptor α subunit (IL-13Rα). In various embodiments,the polypeptides bind IL-2Rβ

_(c) or IL-4Rα

_(c) with a binding affinity of 200 nM or less, 100 nM or less, 50 nM orless or 25 nM or less.

In one aspect, the invention provides non-naturally occurringpolypeptides comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence at least 85%identical to EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence at least 85%identical to YAFNFELI (SEQ ID NO:2);

(d) X4 is a peptide comprising the amino acid sequence at least 85%identical to ITILQSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)). In various embodiments, the polypeptides bind IL-2Rβ

_(c) with a binding affinity of 200 nM or less, 100 nM or less, 50 nM orless or 25 nM or less.

In one aspect, the invention provides non-naturally occurringpolypeptides comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence EHALYDAL (SEQ IDNO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ IDNO:2);

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ IDNO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)). In various embodiments, the polypeptides bind IL-2Rβ

_(c) with a binding affinity of 200 nM or less, 100 nM or less, 50 nM orless or 25 nM or less.

As shown in the examples that follow, the polypeptides of the disclosureare (a) mimetics of IL-2 and interleukin-15 (IL-15) that bind to theIL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)), but have no binding site for IL-2Rα or IL-15Rα, or (b) mimeticsof IL-4 that bind to the IL-4 receptor α

_(c) heterodimer (IL-4Rα

_(c)) or IL-13 receptor α subunit (IL-13Rα) (natural IL-4 and the IL-4mimetics described herein cross-react with IL-13 receptor, forming anIL-4Rα/IL13Rα heterodimer). The designs are hyper-stable, bind to humanand mouse IL-2Rβ

_(c) or IL-4Rα

_(c) with higher affinity than the natural cytokines, and elicitdownstream cell signaling independent of IL-2Rα and IL-15Rα, orindependent of IL-13Rα. The polypeptides can be used, for example, totreat cancer.

The term protein mimetic as used herein refers to a protein thatimitates certain aspects of the function of another protein. The twoproteins typically have different amino acid sequence and/or differentstructures. Provided herein, among other things, are de novo mimetics ofIL-2 and IL-15. The aspects of the function of IL-2 and IL-15 that thesemimetics imitate is the induction of heterodimerization of IL-2Rβ

c, leading to phosphorylation of STAT5. Because IL-2 and IL-15 bothsignal through heterodimerization of IL-2Rβ

c, these mimetics imitate this biological function of both IL-2 andIL-15. These mimetics may be referred to herein as mimetics of IL-2, ofIL-15, or of both IL-2 and IL-15.

Also provided are de novo mimetics of IL-4. These mimetics are capableof imitating certain functions of IL-4. The function of IL-4 that thesemimetics imitate is the induction of heterodimerization of IL-4Rα

_(c) (and/or heterodimerization of IL-4Rα/IL-13Rα).

Native hIL-2 comprises four helices connected by long irregular loops.The N-terminal helix (H1) interacts with both the beta and gammasubunits, the third helix (H3) interacts with the beta subunit, and theC-terminal helix (H4) with the gamma subunit; the alpha subunitinteracting surface is formed by the irregular second helix (H2) and twolong loops, one connecting H1 to H2 and the other connecting H3 and H4.Idealized proteins were designed and produced in which H1, H3 and H4 arereplaced by idealized structural domains, including but not limited tohelices and beta strands (referred to as domains X1, X3 and X4,respectively) displaying an IL-2Rβ

_(c) or IL-4Rα

_(c) interface inspired by H1, H3 and H4, and in which H2 is replacedwith an idealized helix (referred to as domain X2) that offers betterpacking. As shown in the examples, extensive mutational studies havebeen carried out, demonstrating that the amino acid sequence of eachpeptide domain each can be extensively modified without loss of bindingto the IL-2 or IL-4 receptor, and that the domains can be placed in anyorder while retaining binding to the IL-2 or IL-4 receptor. Thepolypeptides may comprise L amino acids and glycine, D-amino acids andglycine, or combinations thereof.

Thus, X1, X2, X3, and X4 may be in any order in the polypeptide; innon-limiting embodiments, the ordering may be X1-X2-X3-X4; X1-X3-X2-X4;X1-X4-X2-X3; X3-X2-X1-X4; X4-X3-X2-X1; X2-X3-X4-X1; X2-X1-X4-X3; etc.

The domains may be separated by amino acid linkers of any length ofamino acid composition. There is no requirement for linkers; in oneembodiment there are no linkers present between any of the domains. Inother embodiments, an amino acid linker may be present between 1, 2, orall 3 junctions between domains X1, X2, X3, and X4. The linker may be ofany length as deemed appropriate for an intended use.

In various embodiments, X1 is a peptide comprising the amino acidsequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO:1. In other embodiments, X3 is a peptidecomprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95% m 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2. In furtherembodiments, X4 is a peptide comprising the amino acid sequence at least25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO:3.

In one embodiment, the polypeptides are IL-2/15 mimetics and (i) X1includes one or both of the following: H at residue 2 and Y at residue5; and/or (ii) X3 includes 1, 2, 3, 4, or all 5 of the following: Y atresidue 1, F at residue 3, N at residue 4, L at residue 7, and I atresidue 8. In a further embodiment, (iii) X4 includes I at residue 8.

In another embodiment, the polypeptides are IL-4 mimetics, and (i) X1includes E at residue 2 and K at residue 5; and (ii) X3 includes F atresidue 1, K at residue 3, R at residue 4, R at residue 7, and N atresidue 8. In a further embodiment, (iii) X4 includes F at residue 8.

In all of these embodiments, X1, X3, and X4 may be any suitable length,meaning each domain may contain any suitable number of additional aminoacids other than the peptides of SEQ ID NOS: 1, 2, and 3, respectively.In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along itslength the peptide LEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is apeptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical along its length to the peptide EDEQEEMANAIITILQSWIFS (SEQ IDNO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 80% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 80% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 80% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 85% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 85% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 85% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 90% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 90% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 90% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 95% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 95% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 95% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence100% identical along its length to the peptide PKKKIQLHAEHALYDALMILNI(SEQ ID NO: 4); X3 is a peptide comprising the amino acid sequence 100%identical along its length to the peptide LEDYAFNFELILEEIARLFESG (SEQ IDNO:5); and X4 is a peptide comprising the amino acid sequence 100%identical along its length to the peptide EDEQEEMANAIITILQSWIFS (SEQ IDNO:6).

In one embodiment, the polypeptides are IL-2/15 mimetics and (i) X1includes 1, 2, 3, 4, or all 5 of the following: L at residue 7, H atresidue 8, H at residue 11, Y at residue 14; M at residue 18; and/or(ii) X3 includes 1, 2, 3, 4, 5, 6, 7, or all 8 of the following: D atresidue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue10, I at residue 11, E at residue 13, or E at residue 14. In a furtherembodiment, (iii) X4 includes I at residue 19.

In one embodiment of IL-2 mimetics, amino acid substitutions relative tothe reference peptide domains (i.e.: SEQ ID NOS: 1, 2, 3, 4, 5, or 6) donot occur at AA residues marked in bold font.

In another embodiment, the polypeptides are IL-4/IL-13 mimetics, and

X1 is a peptide comprising the amino acid sequence at least 25%, 27%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 100% identical along its length to the peptide PKKKIQIMA

SILNI (SEQ ID NO: 8);

X3 is a peptide comprising the amino acid sequence at least 37% 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical along its length the peptide LER

LWGIARLFESG (SEQ ID NO: 9); and

X4 is a peptide comprising the amino acid sequence at least 25%, 27%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 100% identical along its length to the peptide EDEQEEMANA

S (SEQ ID NO: 10).

wherein

(i) X1 includes I at residue 7, T or M at residue 8, E at residue 11, Kat residue 14 and S at residue 18; and

(ii) X3 includes R at residue 3, F at residue 4, K at residue 6, R atresidue 7, R at residue 10, N at residue 11, W at residue 13, and G atresidue 14.

In a further embodiment, (iii) X4 includes F at residue 19.

In one embodiment, amino acid substitutions relative to the referencepeptide domains are conservative amino acid substitutions. As usedherein, “conservative amino acid substitution” means a given amino acidcan be replaced by a residue having similar physiochemicalcharacteristics, e.g., substituting one aliphatic residue for another(such as Ile, Val, Leu, or Ala for one another), or substitution of onepolar residue for another (such as between Lys and Arg; Glu and Asp; orGln and Asn). Other such conservative substitutions, e.g., substitutionsof entire regions having similar hydrophobicity characteristics, areknown. Polypeptides comprising conservative amino acid substitutions canbe tested in any one of the assays described herein to confirm that adesired activity, e.g. antigen-binding activity and specificity of anative or reference polypeptide is retained. Amino acids can be groupedaccording to similarities in the properties of their side chains (in A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers,New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro(P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S),Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu(E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturallyoccurring residues can be divided into groups based on common side-chainproperties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2)neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4)basic: His, Lys, Arg; (5) residues that influence chain orientation:Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutionswill entail exchanging a member of one of these classes for anotherclass. Particular conservative substitutions include, for example; Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into H is; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

In one embodiment, amino acid residues in X1 relative to SEQ ID NO:4 areselected from the group consisting of

Position 01: A F I L M P Q R S W Position 02: A D E G V K Position 03: DE F W K Position 04: D E K N P R W Position 05: D E H I K L M S Position06: A D E G L P S W Q Position 07: D E L Q Y I Position 08: A F H W Y MT Position 09: C F P A Position 10: C D E F K P Position 11: D F H EPosition 12: A D E P S T V Position 13: H I L M P R V W Position 14: F RW Y K Position 15: D E N Y Position 16: A C L M S Position 17: F I L M PR Position 18: G M Q Y S Position 19: I L M P Q V Position 20: A K L M QR S Position 21: G K N P R S W Position 22: D E I K M N W Y

In one embodiment the polypeptides are IL-4 mimetics, and position 7 isI, position 8 is M or T, position 11 is E, position 14 is K, andposition 18 is S.

In another embodiment the polypeptides are IL-2 mimetics, and 1, 2, 3,4, or 5 of the following are not true: position 7 is I, position 8 is Mor T, position 11 is E, position 14 is K, and position 18 is S.

In another embodiment, amino acid residues in X3 relative to SEQ ID NO:5are selected from the group consisting of:

Position 01: A L Position 02: D E G K M T Position 03: D E N Y RPosition 04: C D G T Y F Position 05: A F H S V W Y Position 06: A F I MT V Y K Position 07: D K N S T R Position 08: A C G L M S V F Position09: C H K L R S T V E Position 10: F I L M Y R Position 11: I L N T YPosition 12: F K L M S V Position 13: A D F G I N P Q S T E W Position14: A E F G H S V Position 15: C I L M V W Position 16: A D G S T VPosition 17: H K L N R Position 18: C D G I L Q R T W Position 19: D F MN W Position 20: A C E F G M S Y Position 21: D E G H L M R S T V WPosition 22: A D G K N S Y

In another embodiment, the polypeptides are IL-4/IL-13 mimetics andposition 3 is R, position 4 is F, position 6 is K, position 7 is R,position 10 is R, position 11 is N, position 13 is W, and position 14 isG.

In another embodiment, the polypeptides are IL-2 mimetics and 1, 2, 3,4, 5, 6, 7, or all 8 of the following are not true: position 3 is R,position 4 is F, position 6 is K, position 7 is R, position 10 is R,position 11 is N, position 13 is W, and position 14 is G.

In any of such embodiments, the polypeptide further allows for acysteine at position 17 relative to SEQ ID NO:5 in addition to the aminoacid residues of H, K, L, N and R. Accordingly, amino acid residues inX3 relative to SEQ ID NO:5 can be selected from the group consisting of:

Position 01: A L Position 02: D E G K M T Position 03: D E N Y RPosition 04: C D G T Y F Position 05: A F H S V W Y Position 06: A F I MT V Y K Position 07: D K N S T R Position 08: A C G L M S V F Position09: C H K L R S T V E Position 10: F I L M Y R Position 11: I L N T YPosition 12: F K L M S V Position 13: A D F G I N P Q S T E W Position14: A E F G H S V Position 15: C I L M V W Position 16: A D G S T VPosition 17: H K L N R C Position 18: C D G I L Q R T W Position 19: D FM N W Position 20: A C E F G M S Y Position 21: D E G H L M R S T V WPosition 22: A D G K N S Y

In another embodiment, amino acid residues in X4 relative to SEQ ID NO:6are selected from the group consisting of:

Position 01: D E G K V Position 02: D I M S Position 03: E G H KPosition 04: E G I K Q R S Position 05: A D E G H S V Position 06: C D EG I M Q R T V Position 07: C E L M P R T Position 08: A F L M W Position09: A G L N Q R T Position 10: A C D E F H I W Position 11: I M N S V WPosition 12: I K L S V Position 13: C L M R S T Position 14: I L P T YPosition 15: F G I L M N V Position 16: H K Q R Position 17: C F K S W YPosition 18: K Q T W Position 19: C G N I Position 20: C F G L YPosition 21: A F G H S Y

In another embodiment, the polypeptides are IL-4/IL-13 mimetics andposition 19 is I. In another embodiment, the polypeptides are IL-2mimetics and position 19 is not I.

In any of such embodiments, the polypeptide further allows for acysteine at position 3 relative to SEQ ID NO:6 in addition to the aminoacid residues of E, G, H and K.

Accordingly, amino acid residues in X4 relative to SEQ ID NO:6 can beselected from the group consisting of:

Position 01: D E G K V Position 02: D I M S Position 03: E G H K CPosition 04: E G I K Q R S Position 05: A D E G H S V Position 06: C D EG I M Q R T V Position 07: C E L M P R T Position 08: A F L M W Position09: A G L N Q R T Position 10: A C D E F H I W Position 11: I M N S V WPosition 12: I K L S V Position 13: C L M R S T Position 14: I L P T YPosition 15: F G I L M N V Position 16: H K Q R Position 17: C F K S W YPosition 18: K Q T W Position 19: C G N I Position 20: C F G L YPosition 21: A F G H S Y

As noted herein, domain X2 is a structural domain, and thus any aminoacid sequence that connects the relevant other domains (depending ondomain order) and allows them to fold can be used. The length requiredwill depend on the structure of the protein being made and can be 8amino acids or longer. In one exemplary and non-limiting embodiment, X2is a peptide comprising the amino acid sequence at least 20%, 27%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along itslength to KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7). In a further embodiment,amino acid residues in X2 relative to SEQ ID NO:7 are selected from thegroup consisting of:

Position 01: A H L M R S V K Position 02: A D E Q R S T V W Y Position03: C E G K L N Q R W Position 04: A F G N S T V Y Position 05: A E G IM R V Position 06: C E K L N R V Position 07: A C E I L S T V W Position08: H K L M S T W Y Position 09: A I L M Q S R Position 10: A I M S W YPosition 11: C I K L S V Position 12: C E K L P Q R T Position 13: A D HN W Position 14: A C G I L S T V M Position 15: A E G I K L M R VPosition 16: G H L R S T V Position 17: A I L V Position 18: A C D E G HI K M S Position 19: D E G L N V T

In another embodiment, the polypeptides are IL-4/IL-13 mimetics andposition 11 is I. In another embodiment, the polypeptides are IL-2mimetics and position 11 is not I.

In any of such embodiments, the polypeptide further allows for acysteine at positions 5 or 16 relative to SEQ ID NO:7.

Alternatively, in any of such embodiments, the polypeptide furtherallows for a cysteine at positions 1, 2, 5, 9 or 16 relative to SEQ IDNO:7

Accordingly, amino acid residues in X2 relative to SEQ ID NO:7 can beselected from the group consisting of:

Position 01: A H L M R S V K C Position 02: A D E Q R S T V W Y CPosition 03: C E G K L N Q R W Position 04: A F G N S T V Y Position 05:A E G I M R V C Position 06: C E K L N R V Position 07: A C E I L S T VW Position 08: H K L M S T W Y Position 09: A I L M Q S R C Position 10:A I M S W Y Position 11: C I K L S V Position 12: C E K L P Q R TPosition 13: A D H N W Position 14: A C G I L S T V M Position 15: A E GI K L M R V Position 16: G H L R S T V C Position 17: A I L V Position18: A C D E G H I K M S Position 19: D E G L N V T

In another embodiment, the polypeptide comprises a polypeptide at leastat least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical along its length to the amino acid sequence selected from thegroup consisting of the following polypeptides (i.e.: SEQ ID NOS:11-94,103-184, 190-243, and 245-247). Underlined residues are linkers and areoptional and each residue of the linker, when present, may comprise anyamino acid. For each variant below, two SEQ ID NOS are provided: a firstSEQ ID NO: that includes the linker positions as optional and variable,and a second SEQ ID NO: that lists the sequence as shown below.

G1_neo2_33 H1->H4->STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDLDKAEDIRRNSDQARR H2′->H3EAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 11)STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDLDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 103) G1_neo2_34 H1->H4->STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSCISTGKCDLDKAEDIRRNSDQARR H2′->H3EAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 12)STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSCISTGKCDLDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 104) G1_neo2_35 H1->H4->STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDCDKAEDIRRNSDQARR H2′->H3EAEKRGIDVRDLISNAQVILLEAC (SEQ ID NO: 13)STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDCDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAC (SEQ ID NO: 105) G1_neo2_36 H1->H4->STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELARNLEKVRD H2′->H3EALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 14)STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 106) G1_neo2_37 H1->H4->STKKLQLQAEHFLLDVQMILNESPEPNEELNRCITDAQSWISTGKIDLDRAEECARNLEKVRD H2′->H3EALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 15)STKKLQLQAEHFLLDVQMILNESPEPNEELNRCITDAQSWISTGKIDLDRAEECARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 107) G1_neo2_38 H1->H4->STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSCISTGKCDLDRAEELARNLEKVRD H2′->H3EALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 16)STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSCISTGKCDLDRAEELARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 108) G1_neo2_39 H1->H4->STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELCRNLEKVRD H2′->H3EALKRGIDVRDLVSNACVIALELK (SEQ ID NO: 17)STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELCRNLEKVRDEALKRGIDVRDLVSNACVIALELK (SEQ ID NO: 109) G1_neo2_40 H1->H4->STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSWISTGKIDLDGAKELAKEVEELRQ H2′->H3EAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 18)STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSWISTGKIDLDGAKELAKEVEELRQEAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 110) G1_neo2_41 H1->H4->STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSCISTGKCDLDGAKELAKEVEELRQ H2′->H3EAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 19)STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSCISTGKCDLDGAKELAKEVEELRQEAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 111) G1_neo2_42 H1->H4->STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMAKEAEKIRK H2′->H3EMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 20)STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMAKEAEKIRKEMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 112) G1_neo2_43 H1->H4->STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSCISTGKIDLDNAQEMAKEAEKIRK H2′->H3EMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 21)STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSCISTGKCDLDNAQEMAKEAEKIRKEMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 113) G1_neo2_44 H1->H4->STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMCKEAEKIRK H2′->H3EMEKRGIDVRDLISNICVILLELS (SEQ ID NO: 22)STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMCKEAEKIRKEMEKRGIDVRDLISNICVILLELS (SEQ ID NO: 114) G1_neo2_40_1A H1->H4->STKKTQLLAEHALLDAFMMLNVVPEPNEKLNRIITTMQSWIYTGKIDADGAKELAKEVEELEQE H2′->H3YEKRGIDVEDDASNLKVILLELA (SEQ ID NO: 23)STKKTQLLAEHALLDAFMMLNVVPEPNEKLNRIITTMQSWIYTGKIDADGAKELAKEVEELEQEYEKRGIDVEDDASNLKVILLELA (SEQ ID NO: 115) G1_neo2_40_1B H1->H4->STKKTQLLAEHALLDAHMMLNMLPEPNEKLNRIITTMQSWIHTGKIDGDGAQELAKEVEELEQE H2′->H3YEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 24)STKKTQLLAEHALLDAHMMLNMLPEPNEKLNRIITTMQSWIHTGKIDGDGAQELAKEVEELEQEYEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 116) G1_neo2_40_1C H1->H4->STKKTQLLAEHALLDAFMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQE H2′->H3FEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 25)STKKTQLLAEHALLDAFMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQEFEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 117) G1_neo2_40_1D H1->H4->STKKTQLLAEHALLDALMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQE H2′->H3LEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 26)STKKTQLLAEHALLDALMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQELEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 118) G1_neo2_40_1E H1->H4->STKKTQLLAEHALLDAHMMLNVVPEPNEKLNRIITTMQSWIYTGKIDRDGAQELAKEVEELEQE H2′->H3LEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 27)STKKTQLLAEHALLDAHMMLNVVPEPNEKLNRIITTMQSWIYTGKIDRDGAQELAKEVEELEQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 119) G1_neo2_40_1F H1->H4->STKKTQLLAEHALLDALMMLNLLPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQE H2′->H3HEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 28)STKKTQLLAEHALLDALMMLNLLPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQEHEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 120) G1_neo2_40_1G H1->H4->STKKTQLLAEHALLDAYMMLNMVPEPNEKLNRIITTMQSWILTGKIDSDGAQELAKEVEELEQE H2′->H3LEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 29)STKKTQLLAEHALLDAYMMLNMVPEPNEKLNRIITTMQSWILTGKIDSDGAQELAKEVEELEQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 121) G1_neo2_40_1H H1->H4->STKKTHLLAEHALLDAYMMLNVMPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQE H2′->H3FEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 30)STKKTHLLAEHALLDAYMMLNVMPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQEFEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 122) G1_neo2_40_1I H1->H4->STKKTQLLAEHALLDAYMMLNLVPEPNEKLNRIITTMQSWIFTGKIDADGAQELAIEVEELEQE H2′->H3YEKRGIDVDDYASNLKVILLELA (SEQ ID NO: 31)STKKTQLLAEHALLDAYMMLNLVPEPNEKLNRIITTMQSWIFTGKIDADGAQELAIEVEELEQEYEKRGIDVDDYASNLKVILLELA (SEQ ID NO: 123) G1_neo2_40_1J H1->H4->STKKTQLMAEHALLDAFMMLNVLPEPNEKLNRIITTMQSWIFTGKIDGDDAQELAKEVEELEQE H2′->H3LEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 32)STKKTQLMAEHALLDAFMMLNVLPEPNEKLNRIITTMQSWIFTGKIDGDDAQELAKEVEELEQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 124) G1_neo2_40_ H1->H4->STKKTQLLIEHALLDALDMSRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQQLAKEVEELEQE 1F_H1H2′->H3 HEKRGEDVEDEASNLKVILLELA (SEQ ID NO: 33)STKKTQLLIEHALLDALDMSRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQQLAKEVEELEQEHEKRGEDVEDEASNLKVILLELA (SEQ ID NO: 125) G1_neo2_40_ H1->H4->STKKTQLLLEHALLDALHMRRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQELAKEVEELEQE 1F_H2H2′->H3 HEKRGRDVEDDASNLKVILLELA (SEQ ID NO: 34)STKKTQLLLEHALLDALHMRRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQELAKEVEELEQEHEKRGRDVEDDASNLKVILLELA (SEQ ID NO: 126) G1_neo2_40_ H1->H4->STKKTQLLIEHALLDALNMRKKLPEPNEKLSRIITDMQSWIFTGKIDGDGAQQLAKEVEELEQE 1F_H3H2′->H3 HEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 35)STKKTQLLIEHALLDALNMRKKLPEPNEKLSRIITDMQSWIFTGKIDGDGAQQLAKEVEELEQEHEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 127) G1_neo2_40_ H1->H4->STKKTQLLLEHALLDALHMSRELPEPNEKLNRIITDMQSWIFTGKIDGDGAQDLAKEVEELEQE 1F_H4H2′->H3 HEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 36)STKKTQLLLEHALLDALHMSRELPEPNEKLNRIITDMQSWIFTGKIDGDGAQDLAKEVEELEQEHEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 128) G1_neo2_40_ H1->H4->STKKTQLLIEHALLDALHMSRKLPEPNEKLSRIITTMQSWIFTGKIDGDGAQHLAKEVEELEQE 1F_H5H2′->H3 HEKRGGEVEDEASNLKVILLELA (SEQ ID NO: 37)STKKTQLLIEHALLDALHMSRKLPEPNEKLSRIITTMQSWIFTGKIDGDGAQHLAKEVEELEQEHEKRGGEVEDEASNLKVILLELA (SEQ ID NO: 129) G1_neo2_40_ H1->H4->STKKTQLLIEHALLDALHMKRKLPEPNEKLNRIITNMQSWIFTEKIDGDGAQDLAKEVEELEQE 1F_H6H2′->H3 HEKRGQDVEDYASNLKVILLELA (SEQ ID NO: 38)STKKTQLLIEHALLDALHMKRKLPEPNEKLNRIITNMQSWIFTEKIDGDGAQDLAKEVEELEQEHEKRGQDVEDYASNLKVILLELA (SEQ ID NO: 130) G1_neo2_40_ H1->H4->STEKTQLAAEHALRDALMLKHLLNEPNEKLARIITTMQSWQFTGKIDGDGAQELAKEVEELQQE 1F_M1H2′->H3 HEVRGIDVEDYASNLKVILLHLA (SEQ ID NO: 39)STEKTQLAAEHALRDALMLKHLLNEPNEKLARIITTMQSWQFTGKIDGDGAQELAKEVEELQQEHEVRGIDVEDYASNLKVILLHLA (SEQ ID NO: 131) G1_neo2_40_ H1->H4->STKNTQLAAEDALLDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQE 1F_M2H2′->H3 HEERGIDVEDYASNLKVILLQLA (SEQ ID NO: 40)STKNTQLAAEDALLDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQEHEERGIDVEDYASNLKVILLQLA (SEQ ID NO: 132) G1_neo2_40_ H1->H4->STEKTQHAAEDALRDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQE 1F_M3H2′->H3 HEVRGIDVEDYASNLKVILLQLA (SEQ ID NO: 41)STEKTQHAAEDALRDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQEHEVRGIDVEDYASNLKVILLQLA (SEQ ID NO: 133) G2_neo2_40_ H1->H4->TQKKQQLLAEHALLDALMILNMLKTSSEAVNRMITIAQSWIFTGTSNPEEAKEMIKMAEQAEEE1F_seq02 H2′->H3 ARREGVDTEDYVSNLKVILKEIA (SEQ ID NO: 42)TQKKQQLLAEHALLDALMILNMLKTSSEAVNRMITIAQSWIFTGTSNPEEAKEMIKMAEQAEEEARREGVDTEDYVSNLKVILKEIA (SEQ ID NO: 134) G2_neo2_40_ H1->H4->TTKKYQLLVEHALLDALMMLNLSSESNEKMNRITTTMQSWIFTGTFDPDQAEELAKLVEELREE1F_seq03 H2′->H3 FRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 43)TTKKYQLLVEHALLDALMMLNLSSESNEKMNRIITTMQSWIFTGTFDPDQAEELAKLVEELREEFRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 135) G2_neo2_40_ H1->H4->TTKKIQLLVEHALLDALMILNLSSESNEKLNRIITTLQSWIFRGEIDPDRARELAKLLEEIREE1F_seq04 H2′->H3 MRKRGIDTEDYVSNMIVIIRELA (SEQ ID NO: 44)TTKKIQLLVEHALLDALMILNLSSESNEKLNRIITTLQSWIFRGEIDPDPARELAKLLEEIREEMRKRGIDTEDYVSNMIVIIRELA (SEQ ID NO: 136) G2_neo2_40_ H1->H4->TKKKIQLLAEHVLLDLLMMLNLSSESNEKMNRLITIVQSWIFTGTIDPDQAEEMARWVEELREE1F_seq05 H2′->H3 FRKRGIDTEDYASNVKVILKELS (SEQ ID NO: 45)TKKKIQLLAEHVLLDLLMMLNLSSESNEKMNRLITIVQSWIFTGTIDPDQAEEMAKWVEELREEFRKRGIDTEDYASNVKVILKELS (SEQ ID NO: 137) G2_neo2_40_ H1->H4->TKKKYQLLIEHLLLDALMVLNMSSESNEKLNRIITILQSWIETGTWDPDLAEEMEKLMQEIEEE1F_seq06 H2′->H3 LRRRGIDTEDYMSNMRVIIKELS (SEQ ID NO: 46)TKKKYQLLIEHLLLDALMVLNMSSESNEKLNRIITILQSWIFTGTWDPDLAEEMEKLMQEIEEELRRRGIDTEDYMSNMRVIIKELS (SEQ ID NO: 138) G2_neo2_40_ H1->H4->TKKKLQLLEHLLLDMLMTLNMSSESNEKLNRLITELQSWIFRGEIDPDKAEEMWKIMEEIEKE 1F_seq07H2′->H3 LRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 47)TKKKLQLLVEHLLLDMLMILNMSSESNEKLNRLITELQSWIFRGEIDPDKAEEMWKIMEEIEKELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 139) G2_neo2_40_ H1->H4->TSKKQQLLAEHALLDALMILNISSESSEAVNRAITWLQSWTFKGTVNPDQAEEMRKLAEQTREE1F_seq08 H2′->H3 MRKRGIDTEDYVSNLEVIAKELS (SEQ ID NO: 48)TSKKQQLLAEHALLDALMILNISSESSEAVNPAITWLQSWIFKGTVNPDQAEEMRKLAEQIREEMRKRGIDTEDYVSNLEVIAKELS (SEQ ID NO: 140) G2_neo2_40_ H1->H4->TKKKYQLLIEHLLLDLLMVLNMSSESNEKINRLITWLQSWIFTGTYDPDLAEEMYKILEELREE1F_seq09 H2′->H3 MRERGIDTEDYMSNMRVIVKELS (SEQ ID NO: 49)TKKKYQLLIEHLLLDLLMVLNMSSESNEKINRLITWLQSwIFTGTYDPDLAEEMYKILEELREEMRERGIDTEDYMSNMRVIVKELS (SEQ ID NO: 141) G2_neo2_40_ H1->H4->TKKKWQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWTFTGTYDPDLAEEMKKMMDETEDE1F_seq10 H2′->H3 LRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 50)TKKKWQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFTGTYDPDLAEEMKKMMDEIEDELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 142) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMTLNLSSESNEKLNRIITTMQSWIFTGTIDPDOAEELSKLVEEIREE1F_seq11 H2′->H3 MRKRGIDTEDYVSNLKVILDELS (SEQ ID NO: 51)TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDRDQAEELSKLVEEIREEMRKRGIDTEDYVSNLKVILDELS (SEQ ID NO: 143) G2_neo2_40_ H1->H4->TEKKLQLLVEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGRIDPDKAEELAKLVEELREE1F_seq12 H2′->H3 ARERGIDTEDYVSNLKVILKELS (SEQ ID NO: 52)TEKKLQLLVEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGRIDPDKAEELAKLVEELREEARERGIDTEDYVSNLKVILKELS (SEQ ID NO: 144) G2_neo2_40_ H1->H4->TKKKYQLLMEHLLLDLLMVLNMSSESNEKLNRLITIIQSWIFTGTWDPDKAEEMAKMLKEIEDE1F_seq13 H2′->H3 LRERGIDTEDYMSNMIVIMKELS (SEQ ID NO: 53)TKKKYQLLMEHLLLDLLMVLNMSSESNEKLNRLITIIQSWIFTGTWDRDKAEEMAKMLKEIEDELRERGIDTEDYMSNMIVIMKELS (SEQ ID NO: 145) G2_neo2_40_ H1->H4->TTKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFEGRIDPDQAQELAKLVEELREE1F_seq14 H2′->H3 FRKRGIDTEDYVSNLKVILEELS (SEQ ID NO: 54)TTKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFEGRIDPDQAQELAKLVEELREEFRKRGIDTEDYVSNLKVILEELS (SEQ ID NO: 146) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELAKLVRELREE1F_seq15 H2′->H3 FRKRGIDTEDYASNLEVILRELS (SEQ ID NO: 55)TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELAKLVRELREEFRKRGIDTEDYASNLEVILRELS (SEQ ID NO: 147) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMILNLSSKSNEKLNRIITTMQSWIFNGTIDPDPARELAKLVEEIRDE1F_seq16 H2′->H3 MEKNGIDTEDYVSNLRVILEELA (SEQ ID NO: 56)TKKKIQLLVEHALLDALMILNLSSKSNEKLNRIITTMQSWIFNGTIDPDRARELAKLVEEIRDEMEKNGIDTEDYVSNLKVI LEELA (SEQ ID NO: 148) G2_neo2_40_ H1->H4->TKKKYQLLIEHVLLDLLMLLNLSSESNEKMNRLITILQSWIFTGTYDPDKAEEMAKLLKELREE1F_seq17 H2′->H3 FRERGIDTEDYISNAIVILKELS (SEQ ID NO: 57)TKKKYQLLIEHVLLDLLMLLNLSSESNEKMNRLITILQSWIFTGTYDRDKAEEMAKLLKELREEFRERGIDTEDYISNAIVILKELS (SEQ ID NO: 149) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDPAEELAKLVEELREE1F_seq18 H2′->H3 FRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 58)TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLVEELREEFRKRGIDTEDYASNLKVILKELS (SEQ ID NO; 150) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWITNGTIDPDQARELAKLVEELREE1F_seq19 H2′->H3 FRKRGIDTEDYASNLKVILEELA (SEQ ID NO: 59)TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWTFNGTIDPDQARELAKLVEELREEFRKRGIDTEDYASNLKVILEELA (SEQ ID NO: 151) G2_neo2_40_ H1->H4->TKKKLQLLVEHALLDALMLLNLSSESNEKLNRIITTMQSWIFTGTVDPDQAEELAKLVEEIREE1F_seq20 H2′->H3 LRKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 60)TKKKLQLLVEHALLDALMLLNLSSESNEKLNRIITTMQSWIFTGTVDPDQAEELAKLVEEIREELRKRGIDTEDYVSNLRVILKELS (SEQ ID NO: 152) G2_neo2_40_ H1->H4->TTKKYQLLVEHALLDALMTLNLSSESNEKLNRIITTMQSWIFTGTFDPDQAEELAKLVREIREE1F_seq21 H2′->H3 MRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 61)TTKKYQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWTETGTFDPDQAEELAKLVREIREEMRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 153) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDPAEELAKLVREIREE1F_seq22 H2′->H3 MRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 62)TKKKIQLLVEHALLDALMTLNLSSESNEKLNRITTTMQSWIFTGTIDPDRAEELAKLVREIREEMRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 154) G2_neo2_40_ H1->H4->TKKKYQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFRGEWDPDKAEEWAKILKEIREE1F_seq23 H2′->H3 LRERGIDTEDYMSNAIVIMKELS (SEQ ID NO: 63)TKKKYQLLLEHLLLDLLMILNLSSESNEKLNRLITWLQSWTFRGEWDPDRAEEWAKILKETREELRERGIDTEDYMSNAIVIMKELS (SEQ ID NO: 155) G2_neo2_40_ H1->H4->TDKKLQLLVEHLLLDLLMMLNLSSKSNEKMNRLITIAQSWIFTGKVDPDLAREMIKLLEETEDE1F_seq24 H2′->H3 NRKNGIDTEDYVSNARVIAKELE (SEQ ID NO: 64)TDKKLQLLEHLLLDLLMMLNLSSRSNEKMNRLITTAQSWIFTGKVDPDLAREMIKLLEETEDENRKNGIDTEDYVSNARVIAKELE (SEQ ID NO: 156) G2_neo2_40_ H1->H4->TKKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFTGTIDPDQAEELAKLVEELKEE1F_seq25 H2′->H3 FKKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 65)TKKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWTFTGTIDPDQAEELAKLVEELKEEFKKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 157) G2_neo2_40_ H1->H4->TKKKYQLLIEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGTYDPDKAEELEKLAKEIEDE1F_seq26 H2′->H3 ARERGIDTEDYMSNLRVILKELS (SEQ ID NO: 66)TKKKYQLLIEHALLDALMTLNLWSESNEKLNRIITTMOSWIFTGTYDPDKAEELEKLAKEIEDEARERGIDTEDYMSNLRVILKELS (SEQ ID NO: 158) G2_neo2_40_ H1->H4->TKKKAQLLAEHALLDALMLLNLSSESNERLNRIITWLQSIIFTGTYDPDMVKEAVKLADEIEDE1F_seq27 H2′->H3 MRKRGIDTEDYVSNLRVILQELA (SEQ ID NO: 67)TKKKAQLLAEHALLDALMLLNLSSESNERLNRIITWLQSIIFTGTYDPDMVKEAVKLADEIEDEMRKRGIDTEDYVSNLRVILQELA (SEQ ID NO: 159) G2_neo2_40_ H1->H4->TQKKNQLLAEHLLLDALMVLNQSSESSEVANRIITWAQSWIFEGRYDPNKAEEAKKLAKKLEEE1F_seq28 H2′->H3 MRKRGIDMEDYISNMKVIAEEMS (SEQ ID NO: 68)TQKKNQLLAEHLLLDALMVLNQSSESSEVANRIITWAQSWIFEGRVDPNKAEEAKKLAKKLEEEMRKRGIDMEDYISNMKVIAEEMS (SEQ ID NO: 160) G2_neo2_40_ H3->EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYI1F_seq29 H2′->QSQIFEISERIRETDQEKKEESWKKWQLLLEHALLDVLMLLND(SEQ ID NO: 69) H4->H1EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEISERIRETDQEKKEESWKKWQLLLEHALLDVLMLLND (SEQ ID NO: 161) G2_neo2_40_H1->H3->PEKKRQLLLEHILLDALMLLNLXXXXXXNTESKFEDYISNAEVIAEELAKLMESXXLSDEAEKE1F_seq30 H2′->H4KKIKQWLREVWRIWXXXXWSTLEDKARELLNRIITTIQSQIFY (SEQ ID NO: 70)PEKKRQLLLEHILLDALMLLNLLETNPQNTESKFEDYISNAEVIAEELAKLMESLGLSDEAEKEKKIKQWLREVWRIWSSTNWSTLEDKARELLNRIITTIQSQIFY (SEQ ID NO: 162) G2_neo2_40_H1->H3->PEKKRQLLLEHILLDLLMILNMXXXXXXNTESEMEDYWSNVRVILRELARLMEEXXXKELSELM1F_seq31 H2′->H4ERMRKIVEKIRQIVTXXXXLDTAREWLNRLITWIQSLIPR (SEQ ID NO: 71)PEKKRQLLLEHLLLDLLMILNMIETNRENTESEMEDYWSNVRVILRELARLMEELNYKELSELMERMRKIVEKIRQIVTNNSSLDTAREWLNRLITWIQSLIFR (SEQ ID NO: 163) G2_neo2_40_H1->H3->PEKKRQLLAEHALLDALMLLNIIETNSKNTESKMEDYVSNLEVILTEFKKLAEKLNFSEEAERA1F_seq32 H2′->H4ERMKRWARKAYQMMTLDLSLDKAKEMLNRIITILQSIIFN (SEQ ID NO: 72)PEKKRQLLAEHALLDALMLLNIIETNSKNTESKMEDYVSNLEVILTEFKKLAEKLNFSEEAERAERMKRWARKAYQMMTLDLSLDKAKEMLNRIITILQSIIFN (SEQ ID NO: 164) G2_neo2_40_H1->H3->PEKKRQLLAEHLLLDVLMMLNGNASLKDYASNAQVIADEPRELARELGLTDEAKKAEKITEALE1F_seq33 H2′->H4 RAREWLLNNKDKEKAKEALNRAITIAQSWITN (SEQ ID NO: 73)PEKKRQLLAEHLLLDVLMMLNGNASLKDYASNAQVIADEFRELARELGLTDEAKKAEKIIEALERAKEWLLNNKDKEKAKEALNRAITTAQSWIFN (SEQ ID NO: 165) G2_neo2_40_ H1->H3->PEKKRQLLLEHLLLDLLMTLNMLRTNPKNTESDWEDYMSNIEVIIEELRKLMESLGRSEKAKEW1F_seq34 H2′->H4KRMKQWVRRILEIVKNNSDLEEAKEWLNRLITIVQSEIFE (SEQ ID NO: 74)PEKKRQLLLEHLLLDLLMILNMLRTNPKNIESDWEDYMSNIEVIIEELRKIMESLGRSEKAKEWKRMKQWVRRILEIVKNNSDLEEAKEWLNRLITTVQSEIFE (SEQ ID NO: 166) G2_neo2_40_H1->H3->WEKKRQLLLEHLLLDLLMILNMWRTNPQNTESLMEDYMSNAKVTVEELARMMRSQGLEDKAREW1F_seq35 H2′->H4EEMKKRIEEIRQIIQNNSSKERAKEELNRLITYVOSEIFR (SEQ ID NO: 75)WEKKRQLLLEHLLLDLLMILNMWRTNPQNTESLMEDYMSNAKVIVEELAPMMRSQGLEDKAREWEEMKKRIEEIRQIIQNNSSKERAKEELNRLITYVQSEIFR (SEQ ID NO: 167) G2_neo2_40_H1->H3->PKKKIQLLAEHALLDALMILNIVKTNSQNAEEKLEDYASNVEVILEEIARLMESGDQKDEAEKA1F_seq36 H2′->H4 KRMKEWMKRIKTTASEDEQEEMANRIITLLQSWIFS (SEQ ID NO: 76)PKKKIQLLAEHALLDALMILNIVKTNSQNAEEKLEDYASNVEVILEEIARLMESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIITLLQSWIFS (SEQ ID NO: 168) G2_neo2_40_H1->H3->PEKKRQLLAEHALLDALMILNXXXXXXQNAEEKLEDYMSNVEVIMEEFARMMRXXXXSEEAENA1F_seq37 H2′->H4 ERIKKWVRKASSXXXSEEQREMMNPAITLMQSWIFE (SEQ ID NO: 77)PEKKRQLLAEHALLDALMILNTLQTNPQNAEEKLEDYMSNVEVTMEEFARMMRNGDRSEEAENAERIKKWVRKASSTASSEEQREMMNRAITLMQSWTFE (SEQ ID NO: 169) G2_neo2_40_H1->H3->PEKKRQLLAEHLLLDALMVLNMXXXXXXNTEEKLEDYISNMKVIIKEMIELMRSLXXXEEAEKW1F_seq38 H2′->H4 KEALKAVEKIXXXXDSETARELANRIITLAQSAIFY (SEQ ID NO: 78)PEKKRQLLAEHLLLDALMVLNMLTTNSKNTEEKLEDYISNMKVIIKEMIELMRSLGRLEEAENWKEALKAVEKIGSRMDSETARELANRIITLAQSAIFY (SEQ ID NO: 170) G2_neo2_40_H1->H3->PEKKRQLLAEHALLDALMFLNLXXXXXXQAEEKIEDYASNLRVIAEELARLFENLXXXDEAQKA1F_seq39 H2′->H4 KDIKELAERARSXXSSEKRKEAMNRAITTLQSMIFR (SEQ ID NO: 79)PEKKRQLLAEHALLDALMFLNLVETNPDQAEEKIEDYASNLRVIAEELARLFENLGRLDEAQKAKDIKELAERARSRVSSEKRKEAMNRAITILQSMIFR (SEQ ID NO: 171) G2_neo2_40_H1->H3->PEKKRQLLAEHALLDALMTLNIIRTNSDNTESKLEDYISNLKVILEEIARLMESLGLSDEAEKA1F_seq40 H2′->H4 KEAMRLADKAGSTASEEEKKEAMNRVTTWAQSWIFN (SEQ ID NO: 80)PEKKRQLLAEHALLDALMILNIIRTNSDNTESKLEDYISNLKVILEEIARLMESLGLSDEAEKAKEAMRLADKAGSTASEEEKKEAMNRVITWAQSWIFN (SEQ ID NO: 172) G2_neo2_40_H1->H3->PEKKRQLLAEHALLDALMMLNILRTNPDNAEEKLEDYWSNLIVILREIAKLMESLGLTDEAEKA1F_seq41 H2′->H4 KEAARWAEEARTTASKDQRRELANRIITLLQSWIFS (SEQ ID NO: 81)PEKKRQLLAEHALLDALMMLNILRTNPDNAEEKLEDYWSNLIVILREIAKLMESLGLTDEAEKAKEAARWAEEARTTASRDQRRELANRIITLLQSWIFS (SEQ ID NO: 173) G2_neo2_40_H1->H3->PEKKRQLLAEHLLLDALMILNIIETNEQNAESKLEDYISNAKVILDEFREMARDLGLLDEAKKA1F_seq42 H2′->H4 EKMKRWLEKMRSNASSDERREWANRMITTAQSWIFN (SEQ ID NO: 82)PEKKRQLLAEHLLLDALMILNITETNEQNAESKLEDYISNAKVILDEFREMARDLGLLDEAKKAEKMKRWLEKMRSNASSDERREWANRMITTAQSWTFN (SEQ ID NO: 174) G2_neo2_40_H1->H4->TNKKAQLHAEFALHDALMLLNLSSESNERLNRIITWLQSIIFYGTYDPDMVKEAVKDADEIEDE1F_seq27_S3 H2′->H3 MRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 83)TNKKAQLHAEFALHDALMLLNLSSESNERLNRIITWLQSIIFYGTYDPDMVKEAVKDADEIEDEMRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 245) G2_neo2_40_ H1->H4->TNKEAQLHAEFALYDALMLLNLSSESNERLNRIITWLQSIIFYETYDPDMVKEAVKLADEIEDE1F_seq27_S18 H2′->H3 MRKRKIDTEDYVVNLRLILQELA (SEQ ID NO: 84)TNKEAQLHAEFALYDALMLLNLSSESNERLNRIITWLQSIIFYETYDPDMVKEAVKLADEIEDEMRKRKIDTEDYVVNLRLILQELA (SEQ ID NO: 175) G2_neo2_40_ H1->H4->TKKDAELLAEFALYDALMLLNLSSESNERLNEIITWLQSIIFYGTYDPDMVKEAVKLADEIEDE1F_seq27_S22 H2′->H3 MRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 85)TKKDAELLAEFALYDALMLLNLSSESNERLNEIITWLQSIIFYGTYDPDMVKEAVKLADEIEDEMRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 176) G2_neo2_40_ H1->H4->TNKKAQLHAEFALYDALMLLNLSSESNERLNDIITWLQSIIFTGTYDPDMVKEAVKLADEIEDE1F_seq27_S24 H2′->H3 MRKRKIDTEDYWNLRYILQELA (SEQ ID NO: 86)TNKKAQLHAEFALYDALMLLNLSSESNERLNDIITWLQSIIFTGTYDPDMVKEAVKLADEIEDEMRKRKIDTEDYWNLRYILQELA (SEQ ID NO: 177) G2_neo2_40_ H3->EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYI1F_seq29_S6 H2′->QSQIFEVLHGVGETDQEKKEESWKKWDLLLEHALLDVLMLLND (SEQ ID NO: 87) H4->H1EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEVLHGVGETDQEKKEESWKKWDLLLEHALLDVLMLLND (SEQ ID NO: 178) G2_neo2_40_H3-> EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNELITYI1F_seq29_S7 H2′->QSQIFEVIEREGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 88) H4->H1EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNELITYIQSQIFEVIEREGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 179) G2_neo2_40_H3-> EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYI1F_seq29_S8 H2′->QSQIFEVLEGVGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 89) H4->H1EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEVLEGVGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 180) Neoleukin-H1->H3->PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKA 2/15H2′->H4 KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 90) (i. e.PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAG2_neo2_40_ KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 181)1F_seq36_S11) G2_neo2_40_ H1->H3->PKKKIQLLAEHALFDLLMILNIVKTNSQNAEEKLEDYAYNAGVILEEIARLFESGDQKDEAEKA1F_seq36_S12 H2′->H4KRMKEWMKRIKDTASEDEQEEMANEIITILQSWNFS (SEQ ID NO: 91)PKKKIQLLAEHALFDLLMILNIVKTNSQNAEEKLEDYAYNAGVILEEIARLFESGDQKDEAEKAKRMKEWMKRIKDTASEDEQEEMANEIITILQSWNFS (SEQ ID NO: 182) Neoleukin- H1->H3-PKKKIQLYAEHALYDALMILNIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQKDEAEKA2/15-H8Y- H2′->H4 KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 94)K33E PKKKIQLYAEHALYDALMILNIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 246) Neoleukin-PKKKIQLHAEHALYDALMILNIVKTNSPPAEEK LEDYAFNFELILEEIARLFESGDQKDEAEK 2/15AKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 247) (K32 isconsidered to be a residue of the optional linker in this depictedsequence) IL4_G2_neo2_40_PKKKIQITAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMKE1F_seq36_S11 WMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 92)PKKKIQITAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 183) Neoleukin-4PKKKIQIMAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMIE(i.e. WMKRIKTTASEDEQEEMANAIITILQSWFFS (SEQ ID NO: 93) IL4_G2_neo2_40_PKKKIQIMAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMIE1F_seq36_S11_MIF) WMKRIKTTASEDEQEEMANAIITILQSWFFS (SEQ ID NO: 184)

For each variant below, two SEQ ID NOs are provided: a first SEQ ID NO:that lists the sequence as shown below, and a second SEQ ID NO: thatincludes the linker positions as optional and variable.

>Neoleukin-2/15_R50C (SEQ ID NO: 190)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C (SEQ ID NO: 217)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIACLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C(SEQ ID NO: 191)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C (SEQ ID NO: 218)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFCSG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C(SEQ ID NO: 192)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C (SEQ ID NO: 219)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C(SEQ ID NO: 193)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C (SEQ ID NO: 220)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX CDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C(SEQ ID NO: 194)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C (SEQ ID NO: 221)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KCEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C(SEQ ID NO: 195)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C (SEQ ID NO: 222)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEACKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C(SEQ ID NO: 196)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C (SEQ ID NO: 223)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKCMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C(SEQ ID NO: 197)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKCWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C (SEQ ID NO: 224)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKCWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R73C(SEQ ID NO: 198)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R73C (SEQ ID NO: 225)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKCIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_T77C(SEQ ID NO: 199)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTCASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_T77C (SEQ ID NO: 226)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKTCASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E82C (SEQ ID NO: 200)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E82C (SEQ ID NO: 227)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E85C(SEQ ID NO: 201)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQECMANAIITILQSWIFS* >Neoleukin-2/15_E85C (SEQ ID NO: 228)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQECMANAIITILQSWIFS* >Neoleukin-2/15_R50C_R73C(SEQ ID NO: 202)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C_R73C(SEQ ID NO: 229) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIACLFESG XX KDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_R73C (SEQ ID NO: 203)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_R73C(SEQ ID NO: 230) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFCSG XX KDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_R73C (SEQ ID NO: 204)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_R73C(SEQ ID NO: 231) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_R73C (SEQ ID NO: 205)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_R73C(SEQ ID NO: 232) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX CDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_R73C (SEQ ID NO: 206)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_R73C(SEQ ID NO: 233) PKKKIQLHAEHALYDALMILNIX XXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KCEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_R73C (SEQ ID NO: 207)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_R73C(SEQ ID NO: 234) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEACKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_R73C (SEQ ID NO: 208)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_R73C(SEQ ID NO: 235) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEAEKAKCMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C_E82C (SEQ ID NO: 209)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C_E82C(SEQ ID NO: 236) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIACLFESG XX KDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_E82C (SEQ ID NO: 210)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_E82C(SEQ ID NO: 237) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFCSG XX KDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_E82C (SEQ ID NO: 211)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_E82C(SEQ ID NO: 238) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_E82C (SEQ ID NO: 212)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_E82C(SEQ ID NO: 239) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX CDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_E82C (SEQ ID NO: 213)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_E82C(SEQ ID NO: 240) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KCEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_E82C (SEQ ID NO: 214)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_E82C(SEQ ID NO: 241) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEACKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_E82C (SEQ ID NO: 215)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_E82C(SEQ ID NO: 242) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEAEKAKCMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C_E82C (SEQ ID NO: 216)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKCWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C_E82C(SEQ ID NO: 243) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEAEKAKRMK CWMKRIKT XXXEDCQEEMANAIITILQSWIFS*

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:90, 181, and 247.

In another embodiment, the polypeptide comprises a polypeptide identicalto the amino acid sequence of SEQ ID NO:90, 181, or 247, wherein thepolypeptide (i) does not bind to human or murine IL-2Ralpha, (ii) bindsto human IL2RB with an affinity of about 11.2 nM (iii) binds to murineIL2RB with an affinity of about 16.1 nm (iv) binds to human IL-2Rβ

_(c) with an affinity of about 18.8 nM and (v) binds to murine IL-2Rβ

_(c) with an affinity of about 3.4 nM.

In any of these embodiments of the full length polypeptides, thepolypeptide may be an IL-4/IL-13 mimetic, wherein position 7 is I,position 8 is T or M, position 11 is E, position 14 is K, position 18 isS, position 33 is Q, position 36 is R, position 37 is F, position 39 isK, position 40 is R, position 43 is R, position 44 is N, position 46 isW, and position 47 is G. In a further embodiment, position 68 is I andposition 98 is F.

In any of these embodiments of the full length polypeptides, thepolypeptide may be an IL-2 mimetic, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or all 14 of the following are not true: position 7 isI, position 8 is T or M, position 11 is E, position 14 is K, position 18is S, position 33 is Q, position 36 is R, position 37 is F, position 39is K, position 40 is R, position 43 is R, position 44 is N, position 46is W, and position 47 is G. In a further embodiment, one or both of thefollowing are not true: position 68 is I and position 98 is F.

In one embodiment, the IL-2 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein have a three dimensionalstructure with structural coordinates having a root mean squaredeviation of common residue backbone atoms or alpha carbon atoms of lessthan 2.5 Angstroms, less than 1.5 Angstroms, or less than 1 Angstromwhen superimposed on backbone atoms or alpha carbon atoms of the threedimensional structure of native IL-2.

In another embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein have a three dimensionalstructure with structural coordinates having a root mean squaredeviation of common residue backbone atoms or alpha carbon atoms of lessthan 2.5 Angstroms, less than 1.5 Angstroms, or less than 1 Angstromwhen superimposed on backbone atoms or alpha carbon atoms of a threedimensional structure having the structural coordinates of Table E2.

In a further embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein, when in ternary complexwith the mouse IL-2 receptor β

_(c), have a three dimensional structure wherein the structuralcoordinates of common residue backbone atoms or alpha carbon atoms havea root mean square deviation of less than 2.5 Angstroms, less than 1.5Angstroms, or less than 1 Angstrom when superimposed on backbone atomsor alpha carbon atoms of the three dimensional structure of native IL-2when in ternary complex with the mouse IL-2 receptor β

_(c).

In another embodiment, the IL-4 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein have a three dimensionalstructure with structural coordinates comprising a root mean squaredeviation of common residue backbone atoms or alpha carbon atoms of lessthan 2.5 Angstroms less than 1.5 Angstroms, or less than 1 Angstrom whensuperimposed on backbone atoms or alpha carbon atoms of the threedimensional structure of native IL-4.

In each of these embodiments, the three dimensional structure of thepolypeptide may be determined using computational modeling oralternatively, the three dimensional structure of the polypeptide isdetermined using crystallographically-determined structural data.

In one embodiment of any embodiment or combination of embodimentsdisclosed herein, X1, X2, X3, and X4 are alpha-helical domains. Inanother embodiment, the amino acid length of each of X1, X2, X3 and X4is independently at least about 8, 10, 12, 14, 16, 19, or more aminoacids in length. In other embodiments, the amino acid length of each ofX1, X2, X3 and X4 is independently no more than 1000, 500, 400, 300,200, 100, or 50 amino acids in length. In various further embodiments,the amino acid length of each of X1, X2, X3 and X4 is independentlybetween about 8-1000, 8-500, 8-400, 8-300, 8-200, 8-100, 8-50, 10-1000,10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 12-1000, 12-500, 12-400,12-300, 12-200, 12-100, 12-50, 14-1000, 14-500, 14-400, 14-300, 14-200,14-100, 14-50, 16-1000, 16-500, 16-400, 16-300, 16-200, 16-100, 16-50,19-1000, 19-500, 19-400, 19-300, 19-200, 19-100, or about 19-50 aminoacids in length.

In another embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein, X1 binds to the beta andthe gamma subunit of the human IL-2 receptor. In another embodiment ofthe IL-2 mimetic polypeptides of any embodiment or combination ofembodiments disclosed herein, X2 does not bind to the human IL-2receptor. In another embodiment, of the IL-2 mimetic polypeptides of anyembodiment or combination of embodiments disclosed herein, X3 binds tothe beta subunit of the human IL-2 receptor. In a further embodiment ofthe IL-2 mimetic polypeptides of any embodiment or combination ofembodiments disclosed herein, X4 binds to the gamma subunit of the humanIL-2 receptor. In another embodiment or the IL-2 mimetic polypeptides ofany embodiment or combination of embodiments disclosed herein, thepolypeptide does not bind to the alpha subunit of the human or murineIL-2 receptor. In one embodiment, binding to the receptors is specificbinding as determined by surface plasmon resonance at biologicallyrelevant concentrations. In another embodiment, the IL-2 mimeticpolypeptides of any embodiment or combination of embodiments disclosedherein that bind to the IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)) do so with a binding affinity of 200 nm or less, 100 nm or less,50 nM or less, or 25 nM or less. In a further embodiment of the IL-2mimetic polypeptides of any embodiment or combination of embodimentsdisclosed herein, the polypeptide's affinity for the human and mouseIL-2 receptors is about equal to or greater than that of native IL-2.

In one embodiment of the IL-4 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein that bind to the IL-4receptor α

_(c) heterodimer (IL-4Rα

_(c)) do so with a binding affinity of 200 nm or less, 100 nm or less,50 nM or less, or 25 nM or less. In another embodiment of the IL-4mimetic polypeptides of any embodiment or combination of embodimentsdisclosed herein, the polypeptide's affinity for the human and mouseIL-4 receptors is about equal to or greater than that of native IL-4.

In one embodiment of the IL-2 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein, the polypeptide stimulatesSTAT5 phosphorylation in cells expressing the IL-2 receptor with potencyabout equal to or greater than native IL-2. In another embodiment of theIL-2 mimetic polypeptides of any embodiment or combination ofembodiments disclosed herein, the polypeptide stimulates STAT5phosphorylation in cells expressing the IL-2 receptor with potency aboutequal to or greater than native IL-2 in cells expressing IL-2 receptor β

_(c) heterodimer but lacking the IL-2 receptor α.

In another embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein demonstrate thermalstability about equal to or greater than the thermal stability of nativeIL-2.

In a further embodiment, the polypeptides of any embodiment orcombination of embodiments disclosed herein, the polypeptides maintainor recover at least 70%, 80%, or 90% of their folded structure afterthermal stability testing, and/or maintain or recover at least 80% oftheir ellipticity spectrum after thermal stability testing, and/ormaintain or recover at least 70% or 80% of their activity after thermalstability testing. In one embodiment, such activity is determined by aSTAT5 phosphorylation assay. In another embodiment, thermal stability ismeasured by circular dichroism (CD) spectroscopy at 222 nM. In a furtherembodiment, the thermal stability test comprises heating the polypeptidefrom 25 degrees Celsius to 95 degrees Celsius in a one hour time frame,cooling the polypeptide to 25 degrees Celsius in a 5 minute time frameand monitoring ellipticity at 222 nm.

The polypeptides described herein may be chemically synthesized orrecombinantly expressed (when the polypeptide is genetically encodable).The polypeptides may be linked to other compounds, such as stabilizationcompounds to promote an increased half-life in vivo, including but notlimited to albumin, PEGylation (attachment of one or more polyethyleneglycol chains), HESylation, PASylation, glycosylation, or may beproduced as an Fc-fusion or in deimmunized variants. Such linkage can becovalent or non-covalent. For example, addition of polyethylene glycol(“PEG”) containing moieties may comprise attachment of a PEG grouplinked to maleimide group (“

-MAL”) to a cysteine residue of the polypeptide. Suitable examples of

-MAL are methoxy

-MAL 5 kD; methoxy

-MAL 20 kD; methoxy (

)2-MAL 40 kD; methoxy

(MAL)2 5 kD; methoxy

(MAL)2 20 kD; methoxy

(MAL)2 40 kD; or any combination thereof. See also U.S. Pat. No.8,148,109. In other embodiments, the PEG may comprise branched chainPEGs and/or multiple PEG chains.

In one embodiment, the stabilization compound, including but not limitedto a PEG-containing moiety, is linked at a cysteine residue in thepolypeptide. In another embodiment, the cysteine residue is present inthe X2 domain. In some embodiments, the cysteine residue is present, forexample, in any one of a number of positions in the X2 domain. In somesuch embodiments, the X2 domain is at least 19 amino acids in length andthe cysteine residue is at positions 1, 2, 5, 9 or 16 relative to those19 amino acids. In a further embodiment, the stabilization compound,including but not limited to a PEG-containing moiety, is linked to thecysteine residue via a maleimide group, including but not limited tolinked to a cysteine residue present at amino acid residue 62 relativeto SEQ ID NO:90.

In some aspects, the polypeptide is a Neo-2/15 polypeptide and an aminoacid of Neo-2/15 is mutated to a cysteine residue for attachment of astabilization moiety (e.g., PEG-containing moiety) thereto. In someaspects, the polypeptide is a Neo-2/15 polypeptide and the amino acid atpositions 50, 53, 62, 69, 73, 82, 56, 58, 59, 66, 77, or 85 or acombination thereof relative to SEQ ID NO:90, 181, or 247 is mutated toa cysteine residue for attachment of a stabilization moiety (e.g.,PEG-containing moiety) thereto. Accordingly, in a further embodiment,the polypeptide comprises a polypeptide at least 25%, 27%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical to the full length of the amino acid sequence of SEQ ID NO:90,181, or 247 [Neo-2/15], and wherein one, two, three, four, five, or allsix of the following mutations are present:

R50C;

E53C;

E62C;

E69C;

R73C; and/or

E82C.

In a further embodiment, the polypeptide comprises a polypeptide atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical to the full length of the aminoacid sequence of SEQ ID NO:90, 181, or 247, and wherein one, two, three,four, five, six, seven, eight, nine, ten, eleven, or all twelve of thefollowing mutations are present

D56C;

K58C;

D59C;

R66C;

T77C;

E85C;

R50C;

E53C;

E62C;

E69C;

R73C; and/or

E82C.

In a further embodiment, the polypeptide comprises a polypeptide atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical to the full length of the aminoacid sequence selected from the group consisting of SEQ ID NOS: 190-243.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:190 and 217. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:190.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:191 and 218. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:191.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 192 and 219. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:192.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 193 and 220. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:193.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:194 and 221. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:194.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:195 and 222. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:195.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:196 and 223. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%9, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:196.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:197 and 224. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%9, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:197.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:198 and 225. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%9, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:198.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:199 and 226. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%9, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:199.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:200 and 227. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:200.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:201 and 228. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%9, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:201.

In another embodiment, the polypeptide comprises a polypeptide at least25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 100% identical to the full length of the amino acidsequence selected from the group consisting of SEQ ID NO:195, 207, 214,222, 234, and 241; or wherein the polypeptide comprises a polypeptide atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical to the full length of the aminoacid sequence selected from the group consisting of SEQ ID NO:195, 207,and 214.

In a further embodiment, the polypeptide further comprises a targetingdomain. In this embodiment, the polypeptide can be directed to a targetof interest. The targeting domain may be covalently or non-covalentlybound to the polypeptide. In embodiments where the targeting domain isnon-covalently bound to the polypeptide, any suitable means for suchnon-covalent binding may be used, including but not limited tostreptavidin-biotin linkers.

In another embodiment, the targeting domain, when present, is atranslational fusion with the polypeptide. In this embodiment, thepolypeptide and the targeting domain may directly abut each other in thetranslational fusion or may be linked by a polypeptide linker suitablefor an intended purpose. Exemplary such linkers include, but are notlimited, to those disclosed in WO2016178905, WO2018153865 (inparticular, at page 13), and WO 2018170179 (in particular, at paragraphs[0316]-[0317]). In other embodiments, suitable linkers include, but arenot limited to peptide linkers, such as GGGGG (SEQ ID NO: 95), GSGGG(SEQ ID NO: 96), GGGGGG (SEQ ID NO: 97), GGSGGG (SEQ ID NO: 98),GGSGGSGGGSGGSGSG (SEQ ID NO: 99), GSGGSGGGSGGSGSG (SEQ ID NO: 100),GGSGGSGGGSGGSGGGGSGGSGGGSGGGGS (SEQ ID NO: 101), and [GGGGX]_(n)(SEQ IDNO: 102), where X is Q, E or S and n is 2-5.

The targeting domains are polypeptide domains or small molecules thatbind to a target of interest. In one non-limiting embodiment, thetargeting domain binds to a cell surface protein; in this embodiment,the cell may be any cell type of interest that includes a surfaceprotein that can be bound by a suitable targeting domain. In oneembodiment, the cell surface proteins are present on the surface ofcells selected from the group consisting of tumor cells, tumor vascularcomponent cells, tumor microenvironment cells (e.g. fibroblasts,infiltrating immune cells, or stromal elements), other cancer cells andimmune cells (including but not limited to CD8+ T cells, T-regulatorycells, dendritic cells, NK cells, or macrophages). When the cell surfaceprotein is on the surface of a tumor cell, vascular component cell, ortumor microenvironment cell (e.g. fibroblasts, infiltrating immunecells, or stromal elements), any suitable tumor cell, vascular componentcell, or tumor microenvironment cell surface marker may be targeted,including but not limited to EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN,MUC16, PSMA, TROP2, ROR1, RON, PD-L1, CD47, CTLA-4, CD5, CD19, CD20,CD25, CD37, CD30, CD33, CD40, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3,B7-DC, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, MUC1,folate-binding protein, A33, G250, prostate-specific membrane antigen(PSMA), ferritin, GD2, GD3, GM2, Le^(y), CA-125, CA19-9, epidermalgrowth factor, p185HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR) fibroblastactivation protein, tenascin, a metalloproteinase, endostatin, vascularendothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, Her-2/neu,MAGE A3, p53 nonmutant, NY-ESO-1, MelanA/MART1, Ras mutant, gp100, p53mutant, PRI, bcr-abl, tyronsinase, survivin, PSA, hTERT, a Sarcomatranslocation breakpoint protein, EphA2, PAP, MLL-IAP, AFP, ERG, NA17,PAX3, ALK, androgen receptor, cyclin B 1, polysialic acid, MYCN, RhoC,TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe (animal),CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3,STn, Carbonic anhydrase IX, PAX5, OY-TESL Sperm protein 17, LCK, HMWMAA,AKAP-4, SSX2, XAGE 1, Legumarin, Tie 3, VEGFR2, MAD-CT-1, PDGFR-B,MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3,MUC15, CA6, NAPI2B, TROP2, CLDN6, CLDN16, CLDN18.2, CLorf186, RON, LY6E,FRA, DLL3, PTK7, STRA6, TMPRSS3, TMPRSS4, TMEM238, UPK1B, VTCN1, LIV1,ROR1, and Fos-related antigen 1.

In other embodiments, when the cell surface protein is on the surface ofa tumor cell, vascular component cell, or tumor microenvironment cell(e.g. fibroblasts, infiltrating immune cells, or stromal elements), anysuitable tumor cell, vascular component cell, or tumor microenvironmentcell surface marker may be targeted, including but not limited totargets in the following list:

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM.sub.-001203);(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.-003486);(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM.sub.-012449);(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM.sub.-005823);(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM.sub.-006424);(7) Sema 5b (FLJ10372, KIAA1445, Mm. 42015, SEMA5B, SEMAG, Semaphorin 5bH log, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878);(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628);(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM.sub.-017763);(11) STEAP2 (HGNC.sub.-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,prostate cancer associated gene 1, prostate cancer associated protein 1,six transmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138);(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM.sub.-017636);(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP.sub.-003203 or NM.sub.-003212);(14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr virusreceptor) or Hs. 73792, Genbank accession no. M26004);(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accessionno. NM.sub.-000626);(16) FcRH2 (IFGP4, IRTA4, SPAPlA (SH2 domain containing phosphataseanchor protein 1a), SPAPIB, SPAPIC, Genbank accession no. NM_-030764);(17) HER2 (Genbank accession no. M11730);(18) NCA (Genbank accession no. M18728);(19) MDP (Genbank accession no. BC017023);(20) IL20R.alpha. (Genbank accession no. AF184971);(21) Brevican (Genbank accession no. AF229053);(22) Ephb2R (Genbank accession no. NM_-004442);(23) ASLG659 (Genbank accession no. AX092328);(24) PSCA (Genbank accession no. AJ297436);(25) GEDA (Genbank accession no. AY260763);(26) BAFF-R (Genbank accession no. NP_-443177.1);(27) CD22 (Genbank accession no. NP-001762.1);(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation, Genbank accession No. NP_-001774.1);(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbankaccession No. NP_-001707.1);(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+T lymphocytes, Genbank accessionNo. NP_-002111.1);(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability, Genbank accessionNo. NP_-002552.2);(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accessionNo. NP_-001773.1);(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis, Genbankaccession No. NP_-005573.1);(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation, Genbank accession No.NP_-443170.1); or(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies, Genbank accession No. NP_-112571.1).

In another embodiment, the targeting domain binds to immune cell surfacemarkers. In this embodiment, the target may be cell surface proteins onany suitable immune cell, including but not limited to CD8+ T cells,T-regulatory cells, dendritic cells, NK cells or macrophages. Thetargeting domain may target any suitable immune cell surface marker(whether an endogenous or an engineered immune cell, including but notlimited to engineered CAR-T cells), including but not limited to CD3,CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123,CD254, PD-1, B7-H3, and CTLA-4. In another embodiment, the targetingdomain binds to PD-1, PDL-1, CTLA-4, TROP2, B7-H3, CD33, CD22, carbonicanhydrase IX, CD123, Nectin-4, tissue factor antigen, CD154, B7-H3,B7-H4, FAP (fibroblast activation protein) or MUC16, and/or wherein thetargeting domain binds to PD-1, PDL-1, CTLA-4, TROP2, B7-H3, CD33, CD22,carbonic anhydrase IX, CD123, Nectin-4, tissue factor antigen, CD154,B7-H3, B7-H4, FAP (fibroblast activation protein) or MUC16.

In all these embodiments, the targeting domains can be any suitablepolypeptides that bind to targets of interest and can be incorporatedinto the polypeptide of the disclosure. In non-limiting embodiments, thetargeting domain may include but is not limited to an scFv, a F(ab), aF(ab′)₂, a B cell receptor (BCR), a DARPin, an affibody, a monobody, ananobody, diabody, an antibody (including a monospecific or bispecificantibody); a cell-targeting oligopeptide including but not limited toRGD integrin-binding peptides, de novo designed binders, aptamers, abicycle peptide, conotoxins, small molecules such as folic acid, and avirus that binds to the cell surface.

In another embodiment, the polypeptides include at least one disulfidebond (i.e.: 1, 2, 3, 4, or more disulfide bonds). Any suitable disulfidebonds may be used, such as disulfide bonds linking two differenthelices. In one embodiment, the disulfide bonds include a disulfide bondlinking helix 1 (X1) and helix 4 (X4). The disulfide bond may, forexample, improve the thermal stability of the polypeptide as compared toa substantially similar polypeptide with no disulfide bond linking twodomains together.

The polypeptides and peptide domains of the invention may includeadditional residues at the N-terminus, C-terminus, or both that are notpresent in the polypeptides or peptide domains of the disclosure; theseadditional residues are not included in determining the percent identityof the polypeptides or peptide domains of the disclosure relative to thereference polypeptide. Such residues may be any residues suitable for anintended use, including but not limited to detection tags (i.e.:fluorescent proteins, antibody epitope tags, etc.), adaptors, ligandssuitable for purposes of purification (His tags, etc.), other peptidedomains that add functionality to the polypeptides, etc. Residuessuitable for attachment of such groups may include cysteine, lysine orp-acetylphenylalanine residues or can be tags, such as amino acid tagssuitable for reaction with transglutaminases as disclosed in U.S. Pat.Nos. 9,676,871 and 9,777,070.

In a further aspect, the present invention provides nucleic acids,including isolated nucleic acids, encoding a polypeptide of the presentinvention that can be genetically encoded. The isolated nucleic acidsequence may comprise RNA or DNA. Such isolated nucleic acid sequencesmay comprise additional sequences useful for promoting expression and/orpurification of the encoded protein, including but not limited to polyAsequences, modified Kozak sequences, and sequences encoding epitopetags, export signals, and secretory signals, nuclear localizationsignals, and plasma membrane localization signals. It will be apparentto those of skill in the art, based on the teachings herein, whatnucleic acid sequences will encode the polypeptides of the invention.

In another aspect, the present invention provides recombinant expressionvectors comprising the isolated nucleic acid of any aspect of theinvention operatively linked to a suitable control sequence.“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any control sequences capable ofeffecting expression of the gene product. “Control sequences” operablylinked to the nucleic acid sequences of the invention are nucleic acidsequences capable of effecting the expression of the nucleic acidmolecules. The control sequences need not be contiguous with the nucleicacid sequences, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the nucleicacid sequences and the promoter sequence can still be considered“operably linked” to the coding sequence. Other such control sequencesinclude, but are not limited to, polyadenylation signals, terminationsignals, and ribosome binding sites. Such expression vectors include butare not limited to, plasmid and viral-based expression vectors.

The control sequence used to drive expression of the disclosed nucleicacid sequences in a mammalian system may be constitutive (driven by anyof a variety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive). The expression vector must be replicable in thehost organisms either as an episome or by integration into hostchromosomal DNA. In various embodiments, the expression vector maycomprise a plasmid, viral-based vector (including but not limited to aretroviral vector or oncolytic virus), or any other suitable expressionvector. In some embodiments, the expression vector can be administeredin the methods of the disclosure to express the polypeptides in vivo fortherapeutic benefit. In non-limiting embodiments, the expression vectorscan be used to transfect or transduce cell therapeutic targets(including but not limited to CAR-T cells or tumor cells) to effect thetherapeutic methods disclosed herein.

In a further aspect, the present disclosure provides host cells thatcomprise the recombinant expression vectors disclosed herein, whereinthe host cells can be either prokaryotic or eukaryotic. The cells can betransiently or stably engineered to incorporate the expression vector ofthe invention, using techniques including but not limited to bacterialtransformations, calcium phosphate co-precipitation, electroporation, orliposome mediated-, DEAE dextran mediated-, polycationic mediated-, orviral mediated transfection. (See, for example, Molecular Cloning: ALaboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor LaboratoryPress); Culture of Animal Cells: A Manual of Basic Technique, 2^(nd) Ed.(R.I. Freshney. 1987. Liss, Inc. New York, N.Y.)). A method of producinga polypeptide according to the invention is an additional part of theinvention. The method comprises the steps of (a) culturing a hostaccording to this aspect of the invention under conditions conducive tothe expression of the polypeptide, and (b) optionally, recovering theexpressed polypeptide. The expressed polypeptide can be recovered fromthe cell free extract, but preferably they are recovered from theculture medium.

In a further aspect, the present disclosure provides antibodies thatselectively bind to the polypeptides of the disclosure. The antibodiescan be polyclonal, monoclonal antibodies, humanized antibodies, andfragments thereof, and can be made using techniques known to those ofskill in the art. As used herein, “selectively bind” means preferentialbinding of the antibody to the polypeptide of the disclosure, as opposedto one or more other biological molecules, structures, cells, tissues,etc., as is well understood by those of skill in the art.

In another aspect, the present disclosure provides pharmaceuticalcompositions, comprising one or more polypeptides, nucleic acids,expression vectors, and/or host cells of the disclosure and apharmaceutically acceptable carrier. The pharmaceutical compositions ofthe disclosure can be used, for example, in the methods of thedisclosure described below. The pharmaceutical composition may comprisein addition to the polypeptide of the disclosure (a) a lyoprotectant;(b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent;(e) a stabilizer; (f) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is aTris buffer, a histidine buffer, a phosphate buffer, a citrate buffer oran acetate buffer. The pharmaceutical composition may also include alyoprotectant, e.g. sucrose, sorbitol or trehalose. In certainembodiments, the pharmaceutical composition includes a preservative e.g.benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol,benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride.

The polypeptides, nucleic acids, expression vectors, and/or host cellsmay be the sole active agent in the pharmaceutical composition, or thecomposition may further comprise one or more other active agentssuitable for an intended use.

In a further aspect, the present disclosure provides methods fortreating and/or limiting cancer, comprising administering to a subjectin need thereof a therapeutically effective amount of one or morepolypeptides, nucleic acids, expression vectors, and/or host cells ofthe disclosure, salts thereof, conjugates thereof, or pharmaceuticalcompositions thereof, to treat and/or limit the cancer. When the methodcomprises treating cancer, the one or more polypeptides, nucleic acids,expression vectors, and/or host cells are administered to a subject thathas already been diagnosed as having cancer. As used herein, “treat” or“treating” means accomplishing one or more of the following: (a)reducing the size or volume of tumors and/or metastases in the subject;(b) limiting any increase in the size or volume of tumors and/ormetastases in the subject; (c) increasing survival; (d) reducing theseverity of symptoms associated with cancer; (e) limiting or preventingdevelopment of symptoms associated with cancer; and (f) inhibitingworsening of symptoms associated with cancer.

When the method comprises limiting development of cancer, the one ormore polypeptides, nucleic acids, expression vectors, and/or host cellsare administered prophylactically to a subject that is not known to havecancer, but may be at risk of cancer. As used herein, “limiting” meansto limit development of cancer in subjects at risk of cancer, includingbut not limited to subjects with a family history of cancer, subjectsgenetically predisposed to cancer, subjects that are symptomatic forcancer, etc.

The methods can be used to treat or limit development of any suitablecancer, including but not limited to colon cancer, melanoma, renal cellcancer, head and neck squamous cell cancer, gastric cancer, urothelialcarcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lungcancer, hepatocellular carcinoma, pancreatic cancer, Merkel cellcarcinoma colorectal cancer, acute myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiplemyeloma, ovarian cancer, cervical cancer, and any tumor types selectedby a diagnostic test, such as microsatellite instability, tumormutational burden, PD-L1 expression level, or the immunoscore assay (asdeveloped by the Society for Immunotherapy of Cancer).

The subject may be any subject that has or is at risk of developingcancer. In one embodiment, the subject is a mammal, including but notlimited to humans, dogs, cats, horses, cattle, etc.

In a further aspect, the present disclosure provides methods formodulating an immune response in a subject by administering to a subjecta polypeptide, recombinant nucleic acid, expression vector, recombinanthost cell, or the pharmaceutical composition of the present disclosure.

As used herein, an “immune response” being modulated refers to aresponse by a cell of the immune system, such as a B cell, T cell (CD4or CD8), regulatory T cell, antigen-presenting cell, dendritic cell,monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, orneutrophil, to a stimulus. In some embodiments, the response is specificfor a particular antigen (an “antigen-specific response”), and refers toa response by a CD4 T cell, CD8 T cell, or B cell via theirantigen-specific receptor. In some embodiments, an immune response is aT cell response, such as a CD4+ response or a CD8+ response. Suchresponses by these cells can include, for example, cytotoxicity,proliferation, cytokine or chemokine production, trafficking, orphagocytosis, and can be dependent on the nature of the immune cellundergoing the response. In some embodiments of the compositions andmethods described herein, an immune response being modulated is T-cellmediated.

In some aspects, the immune response is an anti-cancer immune response.In some such aspects, an IL-2 mimetic described herein is administeredto a subject having cancer to modulate an anti-cancer immune response inthe subject.

In some aspects, the immune response is a tissue reparative immuneresponse. In some such aspects, an IL-4 mimetic described here isadministered to a subject in need thereof to modulate a tissuereparative immune response in the subject.

In some aspects, the immune response is a wound healing immune response.In some such aspects, an IL-4 mimetic described here is administered toa subject in need thereof to modulate a wound healing immune response inthe subject.

In some aspects, methods are provided for modulating an immune responseto a second therapeutic agent in a subject. In some such aspects, themethod comprises administering a polypeptide of the present disclosurein combination with an effective amount of the second therapeutic agentto the subject. The second therapeutic agent can be, for example, achemotherapeutic agent or an antigen-specific immunotherapeutic agent.In some aspects, the antigen-specific immunotherapeutic agent compriseschimeric antigen receptor T cells (CAR-T cells). In some aspects, thepolypeptide of the present disclosure enhances the immune response ofthe subject to the therapeutic agent. The immune response can beenhanced, for example, by improving the T cell response (including CAR-Tcell response), augmenting the innate T cell immune response, decreasinginflammation, inhibiting T regulatory cell activity, or combinationsthereof.

In some aspects, a cytokine mimetic of the present invention, e.g., anIL-4 mimetic as described herein, will be impregnated to or otherwiseassociated with a biomaterial and the biomaterial will be introduced toa subject. In some aspects, the biomaterial will be a component of animplantable medical device and the device will be, for example, coatedwith the biomaterial. Such medical devices include, for example,vascular and arterial grafts. IL-4 and/or IL-4 associated biomaterialscan be used, for example, to promote wound healing and/or tissue repairand regeneration.

As used herein, a “therapeutically effective amount” refers to an amountof the polypeptide, nucleic acids, expression vectors, and/or host cellsthat is effective for treating and/or limiting cancer. The polypeptides,nucleic acids, expression vectors, and/or host cells are typicallyformulated as a pharmaceutical composition, such as those disclosedabove, and can be administered via any suitable route, including but notlimited to orally, by inhalation spray, ocularly, intravenously,subcutaneously, intraperitoneally, and intravesicularly in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. In one particular embodiment, thepolypeptides, nucleic acids, expression vectors, and/or host cells areadministered mucosally, including but not limited to intraocular,inhaled, or intranasal administration. In another particular embodiment,the polypeptides, nucleic acids, expression vectors, and/or host cellsare administered orally. Such particular embodiments can be administeredvia droplets, nebulizers, sprays, or other suitable formulations.

Any suitable dosage range may be used as determined by attending medicalpersonnel. Dosage regimens can be adjusted to provide the optimumdesired response (e.g., a therapeutic or prophylactic response). Asuitable dosage range for the polypeptides may, for instance, be 0.1ug/kg-100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight. In someembodiments, the recommended dose could be lower than 0.1 mcg/kg,especially if administered locally. In other embodiments, therecommended dose could be based on weight/m² (i.e. body surface area),and/or it could be administered at a fixed dose (e.g., 0.05-100 mg). Thepolypeptides, nucleic acids, expression vectors, and/or host cells canbe delivered in a single bolus, or may be administered more than once(e.g., 2, 3, 4, 5, or more times) as determined by an attendingphysician.

The polypeptides, nucleic acids, expression vectors, and/or host cellsmade be administered as the sole prophylactic or therapeutic agent, ormay be administered together with (i.e.: combined or separately) one ormore other prophylactic or therapeutic agents, including but not limitedto tumor resection, chemotherapy, radiation therapy, immunotherapy, etc.

Example Computing Environment

FIG. 22 is a block diagram of an example computing network. Some or allof the above-mentioned techniques disclosed herein, such as but notlimited to techniques disclosed as part of and/or being performed bysoftware, the Rosetta software suite, RosettaScripts, PyRosetta, Rosettaapplications, and/or other herein-described computer software andcomputer hardware, can be part of and/or performed by a computingdevice. For example, FIG X1 shows protein design system 102 configuredto communicate, via network 106, with client devices 104 a, 104 b, and104 c and protein database 108. In some embodiments, protein designsystem 102 and/or protein database 108 can be a computing deviceconfigured to perform some or all of the herein described methods andtechniques, such as but not limited to, method 300 and functionalitydescribed as being part of or related to Rosetta. Protein database 108can, in some embodiments, store information related to and/or used byRosetta.

Network 106 may correspond to a LAN, a wide area network (WAN), acorporate intranet, the public Internet, or any other type of networkconfigured to provide a communications path between networked computingdevices. Network 106 may also correspond to a combination of one or moreLANs, WANs, corporate intranets, and/or the public Internet.

Although FIG. 22 only shows three client devices 104 a, 104 b, 104 c,distributed application architectures may serve tens, hundreds, orthousands of client devices. Moreover, client devices 104 a, 104 b, 104c (or any additional client devices) may be any sort of computingdevice, such as an ordinary laptop computer, desktop computer, networkterminal, wireless communication device (e.g., a cell phone or smartphone), and so on. In some embodiments, client devices 104 a, 104 b, 104c can be dedicated to problem solving/using the Rosetta software suite.In other embodiments, client devices 104 a, 104 b, 104 c can be used asgeneral purpose computers that are configured to perform a number oftasks and need not be dedicated to problem solving/using the Rosettasoftware suite. In still other embodiments, part or all of thefunctionality of protein design system 102 and/or protein database 108can be incorporated in a client device, such as client device 104 a, 104b, and/or 104 c.

Computing Environment Architecture

FIG. 23A is a block diagram of an example computing device (e.g.,system) In particular, computing device 200 shown in FIG. 23A can beconfigured to: include components of and/or perform one or morefunctions of some or all of the herein described methods and techniques,such as but not limited to, method 300 and functionality described asbeing part of or related to Rosetta. Computing device 200 may include auser interface module 201, a network-communication interface module 202,one or more processors 203, data storage 204, and protein synthesisdevice 220, all of which may be linked together via a system bus,network, or other connection mechanism 205.

User interface module 201 can be operable to send data to and/or receivedata from external user input/output devices. For example, userinterface module 201 can be configured to send and/or receive data toand/or from user input devices such as a keyboard, a keypad, a touchscreen, a computer mouse, a track ball, a joystick, a camera, a voicerecognition module, and/or other similar devices. User interface module201 can also be configured to provide output to user display devices,such as one or more cathode ray tubes (CRT), liquid crystal displays(LCD), light emitting diodes (LEDs), displays using digital lightprocessing (DLP) technology, printers, light bulbs, and/or other similardevices, either now known or later developed. User interface module 201can also be configured to generate audible output(s), such as a speaker,speaker jack, audio output port, audio output device, earphones, and/orother similar devices.

Network-communications interface module 202 can include one or morewireless interfaces 207 and/or one or more wireline interfaces 208 thatare configurable to communicate via a network, such as network 106 shownin FIG. 22. Wireless interfaces 207 can include one or more wirelesstransmitters, receivers, and/or transceivers, such as a Bluetoothtransceiver, a Zigbee transceiver, a Wi-Fi transceiver, a WiMAXtransceiver, and/or other similar type of wireless transceiverconfigurable to communicate via a wireless network. Wireline interfaces208 can include one or more wireline transmitters, receivers, and/ortransceivers, such as an Ethernet transceiver, a Universal Serial Bus(USB) transceiver, or similar transceiver configurable to communicatevia a twisted pair, one or more wires, a coaxial cable, a fiber-opticlink, or a similar physical connection to a wireline network.

In some embodiments, network communications interface module 202 can beconfigured to provide reliable, secured, and/or authenticatedcommunications. For each communication described herein, information forensuring reliable communications (i.e., guaranteed message delivery) canbe provided, perhaps as part of a message header and/or footer (e.g.,packet/message sequencing information, encapsulation header(s) and/orfooter(s), size/time information, and transmission verificationinformation such as CRC and/or parity check values). Communications canbe made secure (e.g., be encoded or encrypted) and/or decrypted/decodedusing one or more cryptographic protocols and/or algorithms, such as,but not limited to, DES, AES, RSA, Diffie-Hellman, and/or DSA. Othercryptographic protocols and/or algorithms can be used as well or inaddition to those listed herein to secure (and then decrypt/decode)communications.

Processors 203 can include one or more general purpose processors and/orone or more special purpose processors (e.g., digital signal processors,application specific integrated circuits, etc.). Processors 203 can beconfigured to execute computer-readable program instructions 206contained in data storage 204 and/or other instructions as describedherein. Data storage 204 can include one or more computer-readablestorage media that can be read and/or accessed by at least one ofprocessors 203. The one or more computer-readable storage media caninclude volatile and/or non-volatile storage components, such asoptical, magnetic, organic or other memory or disc storage, which can beintegrated in whole or in part with at least one of processors 203. Insome embodiments, data storage 204 can be implemented using a singlephysical device (e.g., one optical, magnetic, organic or other memory ordisc storage unit), while in other embodiments, data storage 204 can beimplemented using two or more physical devices.

Data storage 204 can include computer-readable program instructions 206and perhaps additional data. For example, in some embodiments, datastorage 204 can store part or all of data utilized by a protein designsystem and/or a protein database; e.g., protein designs system 102,protein database 108. In some embodiments, data storage 204 canadditionally include storage required to perform at least part of theherein-described methods and techniques and/or at least part of thefunctionality of the herein-described devices and networks.

In some examples, computing device 200 includes protein synthesis device220. Protein synthesis device can synthesize (or generate polypeptidesbased on input data provided to protein synthesis device 220 usingcommands and/or data provided by processors 203 and/or data storage 204.For example, part or all of the functionality of protein synthesisdevice 220 can be performed by a semi-automated or an automated peptidesynthesizer.

FIG. 23B depicts a network 106 of computing clusters 209 a, 209 b, 209 carranged as a cloud-based server system in accordance with an exampleembodiment. Data and/or software for protein design system 102 can bestored on one or more cloud-based devices that store program logicand/or data of cloud-based applications and/or services. In someexamples, protein design system 102 can be a single computing deviceresiding in a single computing center. In other examples, protein designsystem 102 can include multiple computing devices in a single computingcenter, or even multiple computing devices located in multiple computingcenters located in diverse geographic locations.

In some examples, data and/or software for protein design system 102 canbe encoded as computer readable information stored in tangible computerreadable media (or computer readable storage media) and accessible byclient devices 104 a, 104 b, and 104 c, and/or other computing devices.In some examples, data and/or software for protein design system 102 canbe stored on a single disk drive or other tangible storage media, or canbe implemented on multiple disk drives or other tangible storage medialocated at one or more diverse geographic locations.

FIG. 23B depicts a cloud-based server system in accordance with anexample embodiment. In FIG. 23B, the functions of protein design system102 can be distributed among three computing clusters 209 a, 209 b, and209 c. Computing cluster 209 a can include one or more computing devices200 a, cluster storage arrays 210 a, and cluster routers 211 a connectedby a local cluster network 212 a. Similarly, computing cluster 209 b caninclude one or more computing devices 200 b, cluster storage arrays 210b, and cluster routers 211 b connected by a local cluster network 212 b.Likewise, computing cluster 209 c can include one or more computingdevices 200 c, cluster storage arrays 210 c, and cluster routers 211 cconnected by a local cluster network 212 c.

In some examples, each of the computing clusters 209 a, 209 b, and 209 ccan have an equal number of computing devices, an equal number ofcluster storage arrays, and an equal number of cluster routers. In otherexamples, however, each computing cluster can have different numbers ofcomputing devices, different numbers of cluster storage arrays, anddifferent numbers of cluster routers. The number of computing devices,cluster storage arrays, and cluster routers in each computing clustercan depend on the computing task or tasks assigned to each computingcluster.

In computing cluster 209 a, for example, computing devices 200 a can beconfigured to perform various computing tasks of protein design system102. In one example, the various functionalities of protein designsystem 102 can be distributed among one or more of computing devices 200a, 200 b, and 200 c. Computing devices 200 b and 200 c in computingclusters 209 b and 209 c can be configured similarly to computingdevices 200 a in computing cluster 209 a. On the other hand, in someexamples, computing devices 200 a, 200 b, and 200 c can be configured toperform different functions.

In some examples, computing tasks and stored data associated withprotein design system 102 can be distributed across computing devices200 a, 200 b, and 200 c based at least in part on the processingrequirements of protein design system 102, the processing capabilitiesof computing devices 200 a, 200 b, and 200 c, the latency of the networklinks between the computing devices in each computing cluster andbetween the computing clusters themselves, and/or other factors that cancontribute to the cost, speed, fault-tolerance, resiliency, efficiency,and/or other design goals of the overall system architecture.

The cluster storage arrays 210 a, 210 b, and 210 c of the computingclusters 209 a, 209 b, and 209 c can be data storage arrays that includedisk array controllers configured to manage read and write access togroups of hard disk drives. The disk array controllers, alone or inconjunction with their respective computing devices, can also beconfigured to manage backup or redundant copies of the data stored inthe cluster storage arrays to protect against disk drive or othercluster storage array failures and/or network failures that prevent oneor more computing devices from accessing one or more cluster storagearrays.

Similar to the manner in which the functions of protein design system102 can be distributed across computing devices 200 a, 200 b, and 200 cof computing clusters 209 a, 209 b, and 209 c, various active portionsand/or backup portions of these components can be distributed acrosscluster storage arrays 210 a, 210 b, and 210 c. For example, somecluster storage arrays can be configured to store one portion of thedata and/or software of protein design system 102, while other clusterstorage arrays can store a separate portion of the data and/or softwareof protein design system 102. Additionally, some cluster storage arrayscan be configured to store backup versions of data stored in othercluster storage arrays.

The cluster routers 211 a, 211 b, and 211 c in computing clusters 209 a,209 b, and 209 c can include networking equipment configured to provideinternal and external communications for the computing clusters. Forexample, the cluster routers 211 a in computing cluster 209 a caninclude one or more internet switching and routing devices configured toprovide (i) local area network communications between the computingdevices 200 a and the cluster storage arrays 201 a via the local clusternetwork 212 a, and (ii) wide area network communications between thecomputing cluster 209 a and the computing clusters 209 b and 209 c viathe wide area network connection 213 a to network 106. Cluster routers211 b and 211 c can include network equipment similar to the clusterrouters 211 a, and cluster routers 211 b and 211 c can perform similarnetworking functions for computing clusters 209 b and 209 b that clusterrouters 211 a perform for computing cluster 209 a.

In some examples, the configuration of the cluster routers 211 a, 21 1b, and 211 c can be based at least in part on the data communicationrequirements of the computing devices and cluster storage arrays, thedata communications capabilities of the network equipment in the clusterrouters 211 a, 211 b, and 211 c, the latency and throughput of localnetworks 212 a, 212 b, 212 c, the latency, throughput, and cost of widearea network links 213 a, 213 b, and 213 c, and/or other factors thatcan contribute to the cost, speed, fault-tolerance, resiliency,efficiency and/or other design goals of the moderation systemarchitecture.

Example Methods of Operation

FIG. 24 is a flow chart of an example method 300. Method 300 can becarried out by a computing device, such as computing device 200described in the context of at least FIG. 2A. At least the examples ofmethod 300 mentioned below are discussed above.

Method 300 can begin at block 310, where the computing device candetermine a structure for a plurality of residues of a protein using acomputing device, where the structure of the plurality of residuesprovides a particular receptor binding interface. As will be understoodby the skilled practitioner, the determining of a structure for aplurality of residues of a protein where the structure of the pluralityof residues provides a particular receptor binding interface istypically the identification of the original residues of a nativeprotein that bind to a particular receptor binding interface whereas theplurality of designed residues are identified residues that can bind tothe same receptor binding interface.

At block 320, the computing device can determine a plurality of designedresidues using a mimetic design protocol, where the plurality ofdesigned residues provide the particular receptor binding interface, andwhere the plurality of designed residues differ from the plurality ofresidues.

In some examples, determining the plurality of designed residues usingthe mimetic design protocol can include determining an idealized residueusing a database of idealized residues, where the idealized residue isrelated to a designed residue of the plurality of designed residues. Insome of these examples, determining the idealized residue using thedatabase of idealized residues can include: retrieving one or moreidealized fragments related to the idealized residue from the databaseof idealized residues; and determining the idealized residue byreconstructing the related designed residue using the one or moreidealized fragments. In some of these examples, reconstructing therelated designed residue using the one or more idealized fragments caninclude: reconnecting pairs of the one or more idealized fragments by:use of combinatorial fragment assembly of the pairs of the one or moreidealized fragments; and using Cartesian-constrained backboneminimization to determine whether the pairs of the one or more idealizedfragments link two or more of the plurality of designed residues. Insome of these examples, reconstructing the related designed residueusing the one or more idealized fragments can include: verifying thatoverlapping fragments of the idealized residue are idealized fragmentsusing the database of idealized residues; verifying whether theidealized residue does not clash with a target receptor associated withthe particular receptor binding interface; and after verifying that theidealized residue does not clash with a target receptor associated withthe particular receptor binding interface, determining a most probableamino acid at each position of the idealized residue using the databaseof idealized residues. In some of these examples, determining the firstprotein backbone for the protein by assembling the one or moreconnecting helix structures and the plurality of designed residues overthe plurality of combinations can include: recombining the pairs of theone or more idealized fragments by combinatorially recombining the pairsof the one or more idealized fragments; and determining the firstprotein backbone for the protein using the recombined pairs of the oneor more idealized fragments. In some of these examples, combinatoriallyrecombining the pairs of the one or more idealized fragments can includeranking the pairs of the one or more idealized fragments based on aninterconnection length between idealized fragments of the pairs of theone or more idealized fragments.

In other examples, determining the plurality of designed residues usingthe mimetic design protocol can include: determining an idealizedresidue using one or more parametric equations that represent a shape ofa designed residue of the plurality of designed residues; anddetermining a single fragment that closes the idealized residue with atleast one designed residue of the plurality of designed residues. Insome of these examples, the designed residue can include a helicalstructure, and the one or more parametric equations can include anequation related to phi and psi angles of the helical structure. In someof these examples, the equation related to phi and psi angles of thehelical structure can include one or more terms related to an angularpitch of the phi and psi angles of the helical structure.

At block 330, the computing device can determine one or more connectinghelix structures that connect the plurality of designed residues.

At block 340, the computing device can determine a first proteinbackbone for the protein by assembling the one or more connecting helixstructures and the plurality of designed residues over a plurality ofcombinations.

At block 350, the computing device can design a second protein backbonefor the protein for flexibility and low energy structures based on thefirst protein backbone.

At block 360, the computing device can generate an output related to atleast the second protein backbone. In some examples, generating theoutput related to the second protein backbone for the protein caninclude designing one or more molecules based on the second proteinbackbone for the protein.

In other examples, generating the output related to the second proteinbackbone for the protein can include: generating a synthetic gene forthe protein that is based the second protein backbone for the protein;expressing a particular protein in vivo using the synthetic gene; andpurifying the particular protein. In some of these examples, expressingthe particular protein sequence in vivo using the synthetic gene caninclude expressing the particular protein sequence in one or moreEscherichia coli that include the synthetic gene.

In other examples, generating the output related to the second proteinbackbone for the protein can include generating one or more images thatinclude at least part of the second protein backbone for the protein.

In other examples, the computing device can include a protein synthesisdevice; then, generating the output related to at least the secondprotein backbone for the protein can include synthesizing at least thesecond protein backbone for the protein using the protein synthesisdevice.

In one embodiment, the methods are for designing a protein mimetic, asexemplified herein.

Also included are non-naturally occurring proteins prepared by thecomputational methods described herein. The non-naturally occurringproteins can be cytokines, for example, non-naturally occurring IL-2 orIL-4 mimetics.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of embodiments of the present invention only andare presented in the cause of providing what is believed to be the mostuseful and readily understood description of the principles andconceptual aspects of various embodiments of the invention. In thisregard, no attempt is made to show structural details of the inventionin more detail than is necessary for the fundamental understanding ofthe invention, the description taken with the drawings and/or examplesmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

The above definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition or a dictionary known to those of skill inthe art, such as the Oxford Dictionary of Biochemistry and MolecularBiology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

The above description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure. The description of embodiments of thedisclosure is not intended to be exhaustive or to limit the disclosureto the precise form disclosed. While specific embodiments of, andexamples for, the disclosure are described herein for illustrativepurposes, various equivalent modifications are possible within the scopeof the disclosure, as those skilled in the relevant art will recognize.

All of the references cited herein are incorporated by reference.Aspects of the disclosure can be modified, if necessary, to employ thesystems, functions and concepts of the above references and applicationto provide yet further embodiments of the disclosure. These and otherchanges can be made to the disclosure in light of the detaileddescription.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The above detailed description describes various features and functionsof the disclosed systems, devices, and methods with reference to theaccompanying figures. In the figures, similar symbols typically identifysimilar components, unless context dictates otherwise. The illustrativeembodiments described in the detailed description, figures, and claimsare not meant to be limiting. Other embodiments can be utilized, andother changes can be made, without departing from the spirit or scope ofthe subject matter presented herein. It will be readily understood thatthe aspects of the present disclosure, as generally described herein,and illustrated in the figures, can be arranged, substituted, combined,separated, and designed in a wide variety of different configurations,all of which are explicitly contemplated herein.

With respect to any or all of the ladder diagrams, scenarios, and flowcharts in the figures and as discussed herein, each block and/orcommunication may represent a processing of information and/or atransmission of information in accordance with example embodiments.Alternative embodiments are included within the scope of these exampleembodiments. In these alternative embodiments, for example, functionsdescribed as blocks, transmissions, communications, requests, responses,and/or messages may be executed out of order from that shown ordiscussed, including substantially concurrent or in reverse order,depending on the functionality involved. Further, more or fewer blocksand/or functions may be used with any of the ladder diagrams, scenarios,and flow charts discussed herein, and these ladder diagrams, scenarios,and flow charts may be combined with one another, in part or in whole.

A block that represents a processing of information may correspond tocircuitry that can be configured to perform the specific logicalfunctions of a herein-described method or technique. Alternatively oradditionally, a block that represents a processing of information maycorrespond to a module, a segment, or a portion of program code(including related data). The program code may include one or moreinstructions executable by a processor for implementing specific logicalfunctions or actions in the method or technique. The program code and/orrelated data may be stored on any type of computer readable medium suchas a storage device including a disk or hard drive or other storagemedium.

The computer readable medium may also include non-transitory computerreadable media such as computer-readable media that stores data forshort periods of time like register memory, processor cache, and randomaccess memory (RAM). The computer readable media may also includenon-transitory computer readable media that stores program code and/ordata for longer periods of time, such as secondary or persistent longterm storage, like read only memory (ROM), optical or magnetic disks,compact-disc read only memory (CD-ROM), for example. The computerreadable media may also be any other volatile or non-volatile storagesystems. A computer readable medium may be considered a computerreadable storage medium, for example, or a tangible storage device.Moreover, a block that represents one or more information transmissionsmay correspond to information transmissions between software and/orhardware modules in the same physical device. However, other informationtransmissions may be between software modules and/or hardware modules indifferent physical devices.

Numerous modifications and variations of the present disclosure arepossible in light of the above teachings.

Examples

A computational approach for designing de novo cytokine mimetics isdescribed that recapitulate the functional sites of the naturalcytokines, but otherwise are unrelated in topology or amino acidsequence. This strategy was used to design de novo mimetics of IL-2 andinterleukin-15 (IL-15) ¹⁵ that bind to the IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)) ^(16,17), but have no binding site for IL-2Rα or IL-15Rα. Thedesigns are hyper-stable, bind to human and mouse IL-2Rα

_(c) with higher affinity than the natural cytokines, and elicitdownstream cell signaling independent of IL-2Rα and IL-15Rα. Crystalstructures of an experimentally optimized mimetic, neoleukin-2/15, arevery close to the design model and provide the first structuralinformation on the murine IL-2Rβ

_(c) complex. Neoleukin-2/15 has highly efficacious therapeutic activitycompared to IL-2 in murine models of melanoma and colon cancer, withreduced toxicity and no signs of immunogenicity. This strategy forbuilding hyper-stable de novo mimetics can be readily applied to amultitude of natural cytokines and other signaling proteins, enablingthe creation of superior therapeutic candidates with enhanced clinicalprofiles.

Because of the potent biological activity of natural protein hormonesand cytokines, there have been extensive efforts to improve theirpotential therapeutic efficacy through protein engineering. Such effortshave sought to simplify manufacturing, extend half-life, and modulatereceptor interactions ¹⁸⁻²⁰. However, there are inherent challenges tothe development of a new therapeutic when starting with a naturallyoccurring bioactive protein. First, most natural proteins are onlymarginally stable ²¹⁻²⁵, hence amino acid substitutions aimed atincreasing efficacy can decrease expression or cause aggregation, makingmanufacturing and storage difficult. More substantial changes, such asthe deletion or fusion of functional or targeting domains, are oftenunworkable and can dramatically alter pharmacokinetic properties andtissue penetration ¹⁹. Second, any immune response against theengineered variant may cross-react with the endogenous molecule²⁶⁻³⁵with potentially catastrophic consequences. A computational designapproach was developed to generate analogues of natural proteins withimproved therapeutic properties that circumvent these challenges,focusing effort on engineering de novo cytokine mimetics displayingspecific subsets of the receptor binding interfaces optimal for treatingdisease.

Many cytokines interact with multiple different receptorsubunits^(15,16,36-39) and like most naturally occurring proteins,contain non-ideal structural features that compromise stability but areimportant for function. A computational protocol was developed in whichthe structural elements interacting with the desired receptor subunit(s)are fixed in space, and an idealized globular protein structure is builtto support these elements. Previous efforts were extended usingcombinatorial fragment assembly to support short linear epitopes withparametric construction of disembodied helices coupled withknowledge-based loop closure (FIG. 1a-b ). The approach was tested byattempting to de novo design stable idealized proteins with interactionsurfaces mimicking those of human IL-2 (hIL-2) and human IL-15 (hIL-15)for the human IL-2Rβ

_(c) (hIL-2Rβ

_(c)), but entirely lacking the IL-2 receptor alpha (IL-2Rα) interactionsurface. Previous efforts at removing the alpha interaction region inhIL-2, by either mutation ^(9,44,45) (e.g. F42A mutation of Super-2,also known as H9 ⁹) or PEGylation (e.g. NKTR-214 ^(9,13)) have resultedin markedly reduced stability, binding and/or potency of the cytokine onthe hIL-2Rβ

_(c) receptor while failing to completely eliminate the alphainteraction.

Computational design of IL-2/IL-15 mimetics that bind and activateIL-2Rβ

_(c): Native hIL-2 comprises four helices connected by long irregularloops. The N-terminal helix (H1) interacts with both the beta and gammasubunits of the IL-2 receptor, the third helix (H3) interacts with thebeta subunit, and the C-terminal helix (H4) with the gamma subunit; thealpha subunit interacting surface is formed by the irregular secondhelix (H2) and two long loops, one connecting H1 to H2 and the otherconnecting H3 and H4. An idealized protein was designed thatrecapitulates the interface formed by H1, H3 and H4 with beta and gammaand to replace H2 with a regular helix that offers better packing. Thehelices H1, H3 and H4 (see FIG. 1a ) were used as a template for thebinding site, while helix H2 was reconstructed (H2′) using a databaseoff highly-represented clustered-fragments (see Methods). Pairs ofhelices were connected with loops extracted from the same database (seeFIG. 1b ), the resulting helical hairpins combined into fully connectedbackbones (see FIG. 1c ), and Rosetta ⁴⁶⁻⁴⁸ combinatorial flexiblebackbone sequence design calculations were carried out in the presenceof hIL-2Rβ

_(c) (see Methods). The top four computational Txt use z, 148 fsidesigns and eight single-disulfide stapled variations (see Table S1)were selected for experimental characterization by yeast display (seeMethods). Eight designs were found to bind fluorescently-taggedbeta-gamma chimeric IL-2 receptor at low-nanomolar concentrations. Thebest non-disulfide design (G1_neo2_40) was subjected to site saturationmutagenesis followed by selection and combination of affinity-increasingsubstitutions for the murine IL-2Rβ

γ_(c) (mIL-2Rβ

_(c), see FIG. 10). Optimized designs (were expressed recombinantly inE. coli and found to elicit pSTAT5 signaling in vitro on IL-2-responsivemurine cells at low-nanomolar or even picomolar concentrations (seeTable E1), but had relatively low thermal stability (Tm ˜<45° C., seeFIGS. 14 and 15). To improve stability, the computational designprotocol was repeated starting from the backbone of the highest affinityfirst round design (G1_neo2_40_1F, topology: H1->H4->H2′->H3), couplingthe loop building process with parametric variation in helix length(+/−8 amino acids, see FIG. 1a bottom panel). This second approachimproved the quality of the models by enabling the exploration ofsubstantially more combinations of loops connecting each pair ofhelices. The fourteen best designs of the second generation, along withtwenty-seven Rosetta sequence redesigns of G1_neo2_40_1F (see Table S3),were experimentally characterized and all but one were found to bindIL-2 receptor at low-nanomolar concentrations (FIG. 1d , Table E1, andFIG. 16). The three highest affinity and stability designs (one sequenceredesign and two new mimetics) were subjected to site saturationmutagenesis for mIL-2Rβ

_(c) binding (FIGS. 11-13), followed by selection and combination ofaffinity-increasing substitutions for both human and mouse IL-2Rβ

_(c). The matured designs (see Table S4) showed enhanced binding whileretaining hyper-stability (see Table E1). The top design, neoleukin-2/15(also referred to herein as Neo-2/15), is a 100 residue protein with anew topology and sequence quite different from human or murine IL-2 (29%sequence identity to hIL-2 over 89 residues, and 16% sequence identityto mIL-2 over 76 aligned residues, in structural topology-agnostic basedalignment, see Table E1).

Functional characterization of neoleukin-2/15: Neoleukin-2/15 binds withhigh affinity to human and mouse IL-2Rβ

_(c) (Kd ˜38 nM and −19 nM, respectively), but does not interact withIL-2Rα (FIG. 2a ). The affinities of Neoleukin-2/15 for the human andmouse IL-2 receptors (IL-2Rβ and IL-2Rβ

_(c)) are significantly higher than those of the corresponding nativeIL-2 cytokines. In contrast with native IL-2, Neoleukin-2/15 elicitsIL-2Rα-independent signaling in both human and murine IL-2-responsivecells (FIG. 2b , top), and in murine primary T cells (FIG. 2b , bottom).Neoleukin-2/15 activates IL-2Rα− cells more potently than native humanor murine IL-2 in accordance with its higher binding affinity. Inprimary cells, neoleukin-2/15 is more active on IL-2Rα− cells and lessactive on IL-2Rα+ compared to Super-2, presumably due to its completelack of IL-2Rα binding. Neoleukin-2/15 is hyper-stable (see FIG. 17) anddoes not lose binding affinity for hIL-2Rβ

_(c) following incubation at 80° C. for 2 hours, while hIL-2 and Super-2are completely inactivated after 10 minutes (half-inactivation time=˜4.2min and ˜2.6 min, respectively, FIG. 2c ). Similarly, in ex vivo primarycell cultures, neoleukin-2/15 drove T cell survival effectively afterbeing boiled for 60 minutes at 95° C., while these conditionsinactivated both IL-2 and Super-2 (FIG. 2c , bottom). Thermaldenaturation studies were carried out on many other of the designedmimetics, demonstrating their thermal stability as well (see FIG.14-16). This unprecedented stability for a cytokine-like molecule,beyond eliminating the requirement for cold chain storage, suggests arobustness to mutations (see FIGS. 13 and 18-19), genetic fusions andchemical modification greatly exceeding that of native IL-2, which couldcontribute to the development of improved or new therapeutic properties(see FIG. 7).

Structure of monomeric neoleukin-2/15 and ternary complex with mIL-2Rβ

_(c): The X-ray crystal structure of neoleukin-2/15 was determined andfound it to be very close to the computational design model(r.m.s.d.c_(α)=1.1-1.3 Å for the 6 copies in the asymmetric unit, FIG.3a ). The crystal structure of neoleukin-2/15 in a ternary complex withmurine IL-2Rβ

_(c) (FIG. 3b , Table E2) was solved; this may be the first example inwhich a de novo designed protein enabled the structural determination ofa previously unsolved natural receptor complex. The neoleukin-2/15design model and crystal structure align with the mouse ternary complexstructure with r.m.s.d.c_(α) of 1.27 and 1.29 Å, respectively (FIG. 3c). The order of helices in Neoleukin-2/15 (in IL-2 numbering) isH1->H3->H2′->H4 (see FIGS. 1a and 3 a,d). The H1-H3 loop is disorderedin the ternary complex, but helix H3 is in close agreement with thepredicted structure; there is also an outward movement of helix H4 andthe H2′-H4 loop compared to the monomeric structure (FIG. 3c ).Neoleukin-2/15 interacts with mIL-2Rβ via helices H1 and H3, and with

_(c) via the H1 and H4 helices (FIG. 3c ), and these regions alignclosely with both the computational design model (FIG. 3a ) and themonomeric crystal structure (FIG. 3c ). Structural alignment to thepreviously reported crystal structure of the hIL-2 receptor complex ⁴⁹reveals a close agreement between the helical backbones ofNeoleukin-2/15 and hIL-2 in the binding site, despite the differenttopology of the two proteins (FIG. 3d-e ). Some side chain interactionsbetween neoleukin-2/15 and mIL-2Rβ

_(c) are present in the hIL-2-hIL-2Rβ

complex, while others such as L19Y, arose during the computationaldesign process.

Therapeutic applications of neoleukin-2/15: The clinical use of IL-2 hasbeen mainly limited by toxicity ⁵⁰⁻⁵². Although the interactionsresponsible for IL-2 toxicity in humans are incompletely understood, inmurine models toxicity is T cell independent and ameliorated in animalsdeficient in the IL-2Rα chain (CD25+). Thus, many efforts have beendirected to reengineer IL-2 to weaken interactions with IL-2Rα, butmutations in the CD25 binding site can be highly destabilizing ⁶. Theinherent low stability of IL-2 and its tightly evolved dependence onCD25 have been barriers to the translation of reengineered IL-2compounds. Other efforts have focused on IL-15 ^(53,54), since itelicits similar signaling to IL-2 by dimerizing the IL-2Rβ

_(c) but has no affinity for CD25. However, IL-15 is dependent on transpresentation by the IL-15α (CD215) receptor that is displayed primarilyon antigen-presenting cells and natural killer cells. The low stabilityof native IL-15 and its dependence on trans presentation have also beensubstantial barriers to reengineering efforts ⁵³⁻⁵⁵.

Dose escalation studies on naive mice show that mIL-2 preferentiallyexpands regulatory T cells, consistent with preferential binding toCD25+ cells ^(41,56,57), while neoleukin-2/15 primarily drives expansionof CD8⁺ T cells (FIG. 4a ) and does not induce or minimally inducesexpansion of regulatory T cells only at the highest dose tested.Similarly, in a murine model of airway inflammation, which normallyinduces a small percentage of tissue resident CD8+ T cells,neoleukin-2/15 produces an increase in Thy1.2⁻ CD44⁺ CD8⁺ T cellswithout increasing CD4⁺ Foxp3⁺ antigen-specific Tregs in the lymphoidorgans (FIG. 4b ).

De novo protein design allows the circumvention of the structurallimitations of native cytokines, but there is a possibility of elicitinganti-drug antibodies. To test whether neoleukin-2/15 elicits ananti-drug response, tumor-bearing mice were treated daily withneoleukin-2/15 over a period of 2 weeks, and no evidence of anti-drugantibodies was observed in any of the treated animals (FIG. 4c , leftpanel; a similar lack of immune response was observed for other de novodesign therapeutic candidates ⁴¹). Polyclonal antibodies againstneoleukin-2/15 were produced by vaccinating mice with an inactiveneoleukin-2/15 mutant (K.O. neoleukin) in complete Freund's adjuvant.These polyclonal anti-neoleukin-2/15 antibodies did not cross react withhuman or mouse IL-2 (FIG. 4c ). The absence of binding to native IL-2suggests that even if there is an immune response to neoleukin-2/15,this response is unlikely to cross-react with endogenous IL-2.Furthermore, since the sequence identity between neoleukin-2/15 andhIL-2 is low (<30%, see Table E1), an autoimmune response against hostIL-2 is much more likely with previous engineered hIL-2 variants (e.g.Super-2, see Table E1) which differ from endogenous IL-2 by only a fewmutations.

The therapeutic efficacy of neoleukin-2/15 was tested in the poorlyimmunogenic B16F10 melanoma and the more immunogenic CT26 colon cancermouse models. Single agent treatment with neoleukin-2/15 led todose-dependent delays in tumour growth in both cancer models. In CT26colon cancer, single agent treatment showed improved efficacy to thatobserved for recombinant mIL-2 (FIG. 4d and FIG. 5). In B16F10 melanoma,co-treatment with the anti-melanoma antibody TA99 (anti-TRP1) led tosignificant tumour growth delays, while TA99 treatment alone had littleeffect (FIG. 4e and FIG. 6). In long term survival experiments (8weeks), neoleukin-2/15 in combination with TA99 showed substantiallyreduced toxicity and an overall superior therapeutic effect compared tomIL-2 (FIG. 4e ). Mice treated with the combination mIL-2 and TA99steadily lost weight and their overall health declined to the point ofrequiring euthanasia, whereas little decline was observed with thecombination of neoleukin-2/15 and TA99 (FIG. 4e ). Consistent with atherapeutic benefit, neoleukin-2/15 treatment led to a significantincrease in intratumoral CD8:T_(reg) ratios (see FIG. 4f and FIG. 5),which has been previously correlated with effective antitumor immuneresponses 5′. The increases of CD8:T_(reg) ratios by neoleukin-2/15 aredose and antigen dependent (FIG. 4f ); optimum therapeutic effects wereobtained at higher doses and in combination with other immunotherapies(see FIG. 6). Altogether, these data show that neoleukin-2/15 exhibitsthe predicted homeostatic benefit derived from its IL-2 likeimmunopotentiator activity, but without the adverse effects associatedwith CD25⁺ preferential binding. These enhanced properties andlow-toxicity may allow the routine use of neoleukin-2/15 for otherimmunotherapies where recombinant IL-2 is not broadly used. As anexample of such a use, the potential application of neoleukin-2/15 toenhance CAR-T cell therapy (see FIG. 8) was investigated. NSG miceinoculated with 0.5×10⁶ RAJI tumor cells were left untreated, weretreated with 0.8×10⁶ anti-CD19 CAR-T cells (infused 7 days afterinoculation of tumor cells), or were similarly treated with anti-CD19CAR-T cells plus 20 μg/day of either human IL-2 or neoleukin-2/15 ondays 8-14 after tumor inoculation. As expected, Neoleukin-2/15significantly enhanced the anti-tumor effect of CAR-T cell therapy inthis model, slowing growth of the tumor and extending the survival ofthe mouse (data not shown).

De novo design of protein mimetics has the potential to transform thefield of protein-based therapeutics, enabling the development ofbiosuperior molecules with enhanced therapeutic properties and reducedside-effects, not only for cytokines, but for virtually any biologicallyactive molecule with known or accurately predictable structure. Becauseof the incremental nature of current traditional engineering approaches(e.g. 1-3 amino acid substitutions, chemical modification at a singlesite), most of the shortcomings of the parent molecule are inevitablypassed on to the resulting engineered variants, often in an exacerbatedform. By building mimetics de novo, these shortcomings can be completelyavoided: unlike recombinant IL-2 and engineered variants of hIL-2,neoleukin-2/15 can be solubly expressed in E. coli (see FIG. 17),retains activity at high temperature, does not interact with IL-2Rα andis robust to substantial sequence changes that allow the engineering ofnew functions (FIG. 7). Likely because of the small size and highstability of de novo designed proteins, immunogenicity appears to below, and in contrast to incremental variants of hIL-2, any antibodyresponse to the mimetic is unlikely to cross react with the naturalparent cytokine. Because of their high stability and robustness, andtheir tailored interaction surfaces, designed mimetics are likely to beparticularly powerful in next generation therapeutics which combinedifferent protein functionalities, for example targeted versions ofneoleukin-2/15.

Robust modularity of neoleukin-2/15. Disulfide-stapling andreengineering into an IL-4 mimetic: Neoleukin-2/15 is highly modular,allowing to easily tune its properties, such as increasing its stabilityor modify its binding preference. This modularity and robustness wastaken advantage of by introducing, by computational design, stabilityenhancing single-disulfide staples that preserve the function ofneoleukin-2/15 ⁵⁹. For this, two orthogonal strategies were used. First,a disulfide bridge was introduced by searching pairs of positions withfavorable geometrical arrangements followed by flexible backboneminimization. The final design introduced a single disulfide betweenresidues 38 and 75, which stabilizes helices H3 and H2. In the secondapproach, the N- and C-terminus of neoleukin-2/15 was remodeled to allowthe introduction of a single-disulfide staple that encompasses theentire protein (added sequences CNSN (SEQ ID NO:260) and NFQC (SEQ IDNO:261), for N- and C-termini, respectively after removing terminal Pand S residues, see FIG. 18). Both disulfide stapling strategiesincreased the stability of neoleukin-2/15 (Tm>95° C.), while retainingits sequence and function mostly unaffected (see FIG. 18). Themodularity properties of neoleukin-2/15 were used to modify its bindingpreference. All cytokines in the interleukin-2 family interact with the

_(c) and share a common architecture. Therefore, it was hypothesizedthat neoleukin-2/15 could be transformed into another cytokine mimeticof the IL-2 family by changing only amino acids in the half of thebinding-site that interacts with IL-2Rβ (helices H1 and H3). As proof ofa concept, human interleukin-4 (hIL-4) was chosen as target, since itshares extensive structural homology with IL-2 and has potentialapplications in regenerative medicine ^(60,61) Neo-2/15 was modified tobind to the human IL-4 receptor (comprising IL-4Rα and

_(c)) and not to the human IL-2 receptor (comprising IL-2Rβ and

_(c)) by aligning the Neo-2/15 model into the structure of human IL-4bound to its IL-4 receptor, and mutating 14 residues in Neo-2/15 tomatch the amino-acids of IL-4 at those structural positions that mediateinteractions between IL-4 and IL4r (FIG. 7). Binding was furtheroptimized by directed evolution using random mutagenesis and screeningfor high binding affinity variants, which introduced two additionalamino acid substitutions and modified one of the fourteen originalresidues grafted from the IL-4 protein, thereby creating a new proteinNeoleukin-4 with a total of sixteen mutations from Neoleukin-2/15. Theresulting optimized design, neoleukin-4 (see Table S5), wasrecombinantly expressed and purified from E. coli and tested forbinding. Neoleukin-4 binds with high affinity to IL-4Rα receptor, bindscooperatively to IL-4Rα

_(c) (see FIG. 7), and does not bind with any affinity to the IL-2receptor (data not shown) Neoleukin-4 retains the superior thermostableproperties of neoleukin-2/15 (see FIG. 20b,c ), and binds to the IL-13receptor as expected given the natural cross-reactivity of IL-4 to IL-13receptor (data not shown). Altogether, this shows that neoleukin-2/15 isrobust enough to act as a modular scaffold where significant rationalsequence changes can be introduced to modify its function or physicalproperties in a highly predictable way

Methods

Computational design of de novo cytokine mimetics: The design of de novocytokine mimetics began by defining a the structure of hIL-2 in thequaternary complex with the IL-2Rβ

_(c) receptor as template for the design. After inspection, the residuescomposing the binding-site were defined as hotspots using Rosetta'smetadata (PDBInfoLabels). The structure was feed into the new mimeticdesign protocol that is programmed in PyRosetta, and which canautomatically detect the core-secondary structure elements that composethe target-template and produce the resulting de novo mimetic backboneswith full RosettaScripts compatible information for design. Briefly, themimetic building algorithm works as follows. For the first generation ofdesigns, each of the core-elements was idealized by reconstruction usingloops from a clustered database of highly-ideal fragments (fragment-size4 amino acids). After idealization, the mimetic building protocol aimsto reconnect the idealized elements by pairs in all possiblecombinations. To do this it uses combinatorial fragment assembly ofsequence-agnostic fragments from the database, followed bycartesian-constrained backbone minimization for potential solutions(i.e. where the N- and C-ends of the built fragment are close enough tolink the two secondary structures). After minimization, the solutionsare verified to contain highly ideal fragments (i.e. that everyoverlapping fragment that composes the two connected elements is alsocontained within the database) and no backbone clashes with the target(context) receptor. Passing backbone solutions were then profiled usingthe same database of fragments in order to determine the most probableamino acids at each position (this information was encoded in metadataon the design). Next, solutions for pairs of connected secondarystructures were combinatorially recombined to produce fully connectedbackbones by using graph theory connected components. Since the numberof solutions grows exponentially with each pair of elements, at eachfragment combination step we ranked the designs to favor those withshorter interconnections between pairs of core elements, and kept onlythe top solutions to proceed to the next step. Fully connected solutionswere then profiled by layer (interface, core, non-core-surface,surface), in order to restrict the identities of the possible aminoacids to be layer-compatible. Finally, all the information on hotspots,compatible built-fragment amino acids and layers were combined (hotspothas precedence to amino acid probability, and amino acid probabilitytook precedence to layer). These fully profiled backbones were thenpassed to RosettaScripts for flexible backbone design and filtering (seerosetta-script in Appendix A). For the second generation of designs, twoapproaches were followed. In the first approach, sequence redesigns ofthe best first generation optimized design were executed (G1_neo2_40_1F,see Appendix B). In the second approach new mimetics were engineeredusing G1_neo2_40_1F as the target template. The mimetic design protocolin this second generation was similar to the one described for the firstgeneration, but with two key differences. Firstly, the core-fragmentswere no longer built from fragments, but instead by discoveringparametric equations of repetitive phi and psi angles (omega fixed to180°) that result in repetitive secondary structures that recapitulatedeach of the target helices as close as possible, a “pitch” on the phiand psi angles was allowed every X-amino acids in order to allow thehelices the possibility to have curvature (final parameters: H1:, H2:,H3, H4), the sue of these parametric equations allowed to change thesize of each of the core-elements in the target structure at will(either increase or decrease the size), which was coupled (max/min8.a.a.) with the loop building process, and reductions in the size ofthe core elements were not allowed to remove hotspots from the bindingsite. The second difference in the second generation designs, is thatinstead of reconnecting the secondary structure core-elements we used afragment-size of 7 amino acids, and no combinatorial assembly of morethan one fragment was allowed (i.e. a single fragment has to be able toclose a pair of secondary structures). The rest of the design algorithmwas in essence similar to the one followed in the generation one (seeAppendix C). The Rosetta energy functions used were “talaris2013” and“talaris2014”, for the first and second generation of designs,respectively.

The databases of highly ideal fragments used for the design of thebackbones for the de novo mimetics were constructed with the new Rosettaapplication “kcenters_clustering_of_fragments” using an extensivedatabase of non-redundant publicly available protein structures from theRCSB protein data bank, which was comprised of 16767 PDBs for the 4-merdatabase used for the first generation designs, and 7062 PDBs for the7-mer database used for the second generation designs.

Yeast display: Yeast were transformed with genes encoding the proteinsto be displayed together with linearized pETcon3 vector. The vector waslinearized by 100 fold overdigestion by NdeI and XhoI (New EnglandBiolabs) and then purified by gel extraction (Qiagen). The genesincluded 50 bases of overlap with the vector on both the 5′ and 3′ endssuch that homologous recombination would place the genes in framebetween the AGA2 gene and the myc tag on the vector. Yeast were grown inC-Trp-Ura media prior to induction in SGCAA media as previouslydescribed. 12-18 hours after induction, cells were washed in chilleddisplay buffer (50 mM NaPO₄ pH 8, 20 mM NaCl, 0.5% BSA) and incubatedwith varying concentrations of biotinylated receptor (either human ormurine IL-2Rα, IL-2RD, IL-2

, or human IL-4Rα) while being agitated at 4° C. After approximately 30minutes, cells were washed again in chilled buffer, and then incubatedon ice for 5 minutes with FITC-conjugated anti-c-Myc antibody (1 uL per3×10⁶ cells) and streptavidin-phycoerythrin (1 uL per 100 uL volume ofyeast). Yeast were then washed and counted by flow cytometry (Accuri C6)or sorted by FACS (Sony SH800). For experiments in which the initialreceptor incubation was conducted with a combination of biotinylatedIL-2R

and non-biotinylated IL-4Rα, the non-biotinylated receptor was providedin molar excess.

Mutagenesis and affinity maturation: For error-prone PCR basedmutagenesis, the design to be mutated was cloned into pETcon3 vector andamplified using the MutaGene II mutagenesis kit (Invitrogen) permanufacturer's instructions to yield a mutation frequency ofapproximately 1% per nucleotide. 1 μg of this mutated gene waselectroporated into EBY100 yeast together with 1 μg of linearizedpETcon3 vector, with a transformation efficiency on the order of 10⁸.The yeast were induced and sorted multiple times in succession withprogressively decreasing concentrations of receptor until convergence ofthe population. The yeast were regrown in C-Trp-Ura media between eachsort.

Site-saturation mutagenesis (SSM) libraries were constructed fromsynthetic DNA from Genscript. For each amino acid on each designtemplate, forward primers and reverse primers were designed such thatPCR amplification would result in a 5′ PCR product with a degenerate NNKcodon and a 3′ PCR product, respectively. Amplification of “left” and“right” products by COF and COR primers yielded a series of templateproducts each consisting of a degenerate NNK codon at a differentresidue position. For each design, these products were pooled to yieldthe SSM library. SSM libraries were transformed by electroporation intoconditioned Saccharomyces cerevisiae strain EBY100 cells, along withlinearized pETCON3 vector, using the protocol previously described byBenatuil et al.

Combinatorial libraries were constructed from synthetic DNA fromGenscript containing ambiguous nucleotides and similarly transformedinto linearized pETCON3 vector.

Protein expression: Genes encoding the designed protein sequences weresynthesized and cloned into pET-28b(+) E. coli plasmid expressionvectors (GenScript, N-terminal 6×His tag and thrombin cleavage site).Plasmids were then transformed into chemically competent E. coli Lemo21cells (NEB). Protein expression was performed using Terrific Broth and Msalts, cultures were grown at 37° C. until OD⁶⁰⁰ reached approximately0.8, then expression was induced with 1 mM of isopropylβ-D-thiogalactopyranoside (IPTG), and temperature was lowered to 18° C.After expression for approximately 18 hours, cells were harvested andlysed with a Microfluidics M110P microfluidizer at 18,000 psi, then thesoluble fraction was clarified by centrifugation at 24,000 g for 20minutes. The soluble fraction was purified by Immobilized Metal AffinityChromatography (Qiagen) followed by FPLC size-exclusion chromatography(Superdex 75 10/300 GL, GE Healthcare). The purified neoleukin-2/15 wascharacterized by Mass Spectrum (MS) verification of the molecular weightof the species in solution (Thermo Scientific), SizeExclusion—MultiAngle Laser Light Scattering (SEC-MALLS) in order toverify monomeric state and molecular weight (Agilent, Wyatt), SDS-PAGE,and endotoxin levels (Charles River).

Human and mouse IL-2 complex components including hIL-2 (a.a. 1-133),hIL-2Rα (a.a. 1-217), hIL-2Rβ (a.a. 1-214) hIL-2Rγ (a.a. 1-232), mIL-2(a.a. 1-149), mIL-2Rα ectodomain (a.a. 1-213), mIL-2Rβ ectodomain (a.a.1-215), and mγ_(c) ectodomain (a.a. 1-233) were secreted and purifiedusing a baculovirus expression system, as previously described ^(17,49)All proteins were purified to >98% homogeneity with a Superdex 200sizing column (GE Healthcare) equilibrated in HBS. Purity was verifiedby SDS-PAGE analysis. For expression of biotinylated human IL-2 andmouse IL-2 receptor subunits, proteins containing a C-terminal biotinacceptor peptide (BAP)-LNDIFEAQKIEWHE (SEQ ID NO:262) were expressed andpurified as described via Ni-NTA affinity chromatography and thenbiotinylated with the soluble BirA ligase enzyme in 0.5 mM Bicine pH8.3, 100 mM ATP, 100 mM magnesium acetate, and 500 mM biotin (Sigma).Excess biotin was removed by size exclusion chromatography on a Superdex200 column equilibrated in HBS.

Neoleukin-2 crystal and co-crystal structures: C-terminally 6×His-taggedendoglycosidase H (endoH) and murine IL-2Rβ and IL-2Rγ were expressedseparately in Hi-five cells using a baculovirus system as previouslydescribed. IL-2Rγ was grown in the presence of 5 μM kifunensin. Afterapproximately 72 hours, the secreted proteins were purified from themedia by passing over a Ni-NTA agarose column and eluted with 200 mMimidazole in HBS buffer (150 mM NaCl, 10 mM HEPES pH 7.3). EndoH wasexchanged into HBS buffer by diafiltration. mIL-2Rγ was deglycosylatedby overnight incubation with 1:75 (w/w) endoH. mIL-2Rβ and mIL-2Rγ werefurther purified and buffer exchanged by FPLC using an S200 column (GELife Sciences).

Monomeric neoleukin-2/15 was concentrated to 12 mg/ml and crystallizedby vapor diffusion from 2.4 M sodium malonate pH 7.0, and crystals wereharvested and flash frozen without further cryoprotection. Crystalsdiffracted to 2.0 Å resolution at Stanford Synchrotron RadiationLaboratory beamline 12-2 and were indexed and integrated using XDS(Kabsch, 2010). The space group was assigned with Pointless (Evans,2006), and scaling was performed with Aimless (Evans and Murshudov,2013) from the CCP4 suite (Winn et al., 2013). Our predicted model wasused as a search ensemble to solve the structure by molecularreplacement in Phaser (McCoy et al., 2007), with six protomers locatedin the asymmetric unit. After initial rebuilding with Autobuild(Terwilliger et al., 2008), iterative cycles of manual rebuilding andrefinement were performed using Coot (Emsley et al., 2010) and Phenix(Adams et al., 2010).

To crystallize the ternary neoleukin:mIL-2Rβ:mIL-2Rγ complex, the threeproteins were combined in equimolar ratios, digested overnight with1:100 (w/w) carboxypeptidases A and B to remove purification tags, andpurified by FPLC using an S200 column; fractions containing all threeproteins were pooled and concentrated to 20 mg/ml. Initial needlelikemicrocrystals were formed by vapor diffusion from 0.1 M imidazole pH8.0, 1 M sodium citrate and used to prepare a microseed stock forsubsequent use in microseed matrix screening (MMS, (D'Arcy et al.,2014)). After a single iteration of MMS, crystals grown in the sameprecipitant were cryoprotected with 30% ethylene glycol, harvested anddiffracted anisotropically to 3.4 Å×3.8 Å×4.1 Å resolution at AdvancedPhoton Source beamline 231D-B. The structure was solved by molecularreplacement in Phaser using the human IL-2Rβ and IL-2Rγ structures (pdbID 2B5I) as search ensembles. This produced an electron density map intowhich two poly-alanine alpha helices could be manually built. Followingrigid body refinement in Phenix, electron density for the two unmodeledalpha helices, along with the BC loop and some aromatic side chains,became visible, allowing docking of the monomeric neoleukin. Two furtheriterations of MMS and use of an additive screen (Hampton Research)produced crystals grown by vapor diffusion using 150 nl of protein, 125nl of well solution containing 0.1 M Tris pH 7.5, 5% dextran sulfate,2.1 M ammonium sulfate and 25 nl of microseed stock containing 1.3 Mammonium sulfate, 50 mM Tris pH 7.5, 50 mM imidazole pH 8.0, 300 mMsodium citrate. Crystals cryoprotected with 3 M sodium malonate wereflash frozen and diffracted anisotropically to 2.5 Å×3.7 Å×3.8 Å atAdvanced Light Source beamline 5.0.1. After processing the data withXDS, an elliptical resolution limit was applied using the STARANISOserver (Bruhn et al., 2017). Rapid convergence of the model was obtainedby refinement against these reflections using TLS and target restraintsto the higher resolution human receptor (PDB id 2B5I) and neoleukin-2/15structures in Buster (Smart et al., 2012; Bricogne et al., 2016), withmanual rebuilding in Coot, followed by a final round of refinement inPhenix with no target restraints. Structure figures were prepared withPyMol (Schrodinger, L L C. 2010. The PyMOL Molecular Graphics System,Version 2.1.0). Software used in this project was installed andconfigured by SBGrid (Morin et al., 2013).

Cell Lines: Unmodified YT-1⁶⁴ and IL-2Rα⁺ YT-1 human natural killercells⁶⁵ were cultured in RPMI complete medium (RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 2 mM L-glutamine, minimumnon-essential amino acids, sodium pyruvate, 25 mM HEPES, andpenicillin-streptomycin [Gibco]). CTLL-2 cells purchased from ATCC werecultured in RPMI complete with 10% T-STIM culture supplement with ConA(Corning). All cells were maintained at 37° C. in a humidifiedatmosphere with 5% CO₂. The subpopulation of YT-1 cells expressingIL-2Rα was purified via magnetic selection as described previously ¹⁷.Enrichment and persistence of IL-2Rα expression was monitored byanalysis of PE-conjugated anti-human IL-2Rα (Biolegend) antibody bindingon an Accuri C6 flow cytometer (BD Biosciences).

Circular dichroism (CD): Far-ultraviolet CD measurements were carriedout with an AVIV spectrometer model 420 in PBS buffer (pH 7.4) in a 1 mmpath-length cuvette with protein concentration of ˜0.20 mg/ml (unlessotherwise mentioned in the text). Temperature melts where from 25 to 95°C. and monitored absorption signal at 222 nm (steps of 2° C./min, 30 sof equilibration by step). Wavelength scans (195-260 nm) were collectedat 25° C. and 95° C., and again at 25° C. after fast refolding (˜5 min).

Binding studies: Surface plasmon resonance (SPR): For IL-2 receptoraffinity titration studies, biotinylated human or mouse IL-2Rα, IL-2Rβ,and IL-2Rγ receptors were immobilized to streptavidin-coated chips foranalysis on a Biacore T100 instrument (GE Healthcare). An irrelevantbiotinylated protein was immobilized in the reference channel tosubtract non-specific binding. Less than 100 response units (RU) of eachligand was immobilized to minimize mass transfer effects. Three-foldserial dilutions of hIL-2, mIL-2, Super-2, or engineered IL-2 mimeticswere flowed over the immobilized ligands for 60 s and dissociation wasmeasured for 240 s. For IL-2R3ye binding studies, saturatingconcentrations of hIL-2Rβ (3 uM) or mIL-2Rβ M (5 uM) were added to theindicated concentrations of hIL-2 or mIL-2, respectively. Surfaceregeneration for all interactions was conducted using 15 s exposure to 1M MgCl2 in 10 mM sodium acetate pH 5.5. SPR experiments were carried outin HBS-P+ buffer (GE Healthcare) supplemented with 0.2% bovine serumalbumin (BSA) at 25° C. and all binding studies were performed at a flowrate of 50 L/min to prevent analyte rebinding. Data was visualized andprocessed using the Biacore T100 evaluation software version 2.0 (GEHealthcare). Equilibrium titration curve fitting and equilibrium bindingdissociation (KD) value determination was implemented using GraphPadPrism assuming all binding interactions to be first order. Biolayerinterferometry: binding data were collected in a Octet RED96 (ForteBio,Menlo Park, Calif.) and processed using the instrument's integratedsoftware using a 1:1 binding model. Biotinylated target receptors,either human or murine IL-2Rα, IL-2Rβ, IL-2

, or human IL-4Rα, were functionalized to streptavidin coated biosensors(SA ForteBio) at 1 μg/ml in binding buffer (10 mM HEPES [pH 7.4], 150 mMNaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk) for 300seconds. Analyte proteins were diluted from concentrated stocks intobinding buffer. After baseline measurement in binding buffer alone, thebinding kinetics were monitored by dipping the biosensors in wellscontaining 100 nM of the designed protein (association) and then dippingthe sensors back into baseline wells (dissociation). For bindingexperiments in which either IL-2Rβ or IL-4Rα were supplemented insolution while IL-2

was bound to the sensor, the supplemental proteins were provided in 2.5fold molar excess

STAT5 phosphorylation studies: In vitro studies: Approximately 2×10⁵YT-1, IL-2Rα⁺ YT-1, or CTLL-2 cells were plated in each well of a96-well plate and re-suspended in RPMI complete medium containing serialdilutions of hIL-2, mIL-2, Super-2, or engineered IL-2 mimetics. Cellswere stimulated for 15 min at 37° C. and immediately fixed by additionof formaldehyde to 1.5% and 10 min incubation at room temperature.Permeabilization of cells was achieved by resuspension in ice-cold 100%methanol for 30 min at 4° C. Fixed and permeabilized cells were washedtwice with FACS buffer (phosphate-buffered saline [PBS] pH 7.2containing 0.1% bovine serum albumin) and incubated with Alexa Fluor®647-conjugated anti-STAT5 pY694 (BD Biosciences) diluted in FACS bufferfor 2 hours at room temperature. Cells were then washed twice in FACSbuffer and MFI was determined on a CytoFLEX flow cytometer(Beckman-Coulter). Dose-response curves were fitted to a logistic modeland half-maximal effective concentration (EC₅₀ values) were calculatedusing GraphPad Prism data analysis software after subtraction of themean fluorescence intensity (MFI) of unstimulated cells andnormalization to the maximum signal intensity. Experiments wereconducted in triplicate and performed three times with similar results.Ex vivo studies: Spleens and lymph nodes were harvested from wild-typeC57BL/6J or B6; 129S4-Il2ra^(tm1Dw) (CD25KO) mice purchased from TheJackson Laboratory and made into a single cell suspension in sort buffer(2% Fetal Calf Serum in pH 7.2 phosphate-buffered saline). CD4+ T cellswere enriched through negative selection by staining the cell suspensionwith biotin-conjugated anti-B220, CD8, NK1.1, CD11b, CD11c, Ter119, andCD19 antibodies at 1:100 for 30 min on ice. Following a wash with sortbuffer, anti-biotin MicroBeads (Miltenyi Biotec) were added to the cellsuspension at 20 μL per 10⁷ total cells and incubated on ice for 20minutes. Cells were washed, resuspended and negative selection was thenperformed using EasySep Magnets (STEMCELL Technologies). Approximately1×10⁵ enriched cells were added to each well of a 96-well plate in RPMIcomplete medium with 5% FCS with 10-fold serial dilutions of mL-2,Super-2, or Neoleukin-2/15. Cells were stimulated for 20 minutes at 37°C. in 5% CO₂, fixed with 4% PFA and incubated for 30 minutes at 4° C.Following fixation, cells were harvested and washed twice with sortbuffer and again fixed in 500 μL 90% ice-cold methanol in dH₂O for 30minutes on ice for permeabilization. Cells were washed twice withPerm/Wash Buffer (BD Biosciences) and stained with anti-CD4-PerCP inPerm/Wash buffer (1:300), anti-CD44-Alexa Fluor 700 (1:200),anti-CD25-PE-Cy7 (1:200), and 5 μL per sample of anti-pSTAT5-PE pY694for 45 min at room temperature in the dark. Cells were washed withPerm/Wash and re-suspended in sort buffer for analysis on a BD LSR IIflow cytometer (BD Biosciences).

In vivo murine airway inflammation experiments: C57BL/6J were purchasedfrom The Jackson Laboratory. Mice were inoculated intranasally with 20μL of whole house dust mite antigen (Greer) resuspended in PBS to atotal of 23 μg Derp1 per mouse. From Days 1-7, mice were given a dailyintraperitoneal injection of 20 μg mIL-2 in sterile PBS (pH 7.2), amolar equivalent of Neoleukin-2/15 in sterile PBS, or no injection. OnDay 8, circulating T cells were intravascularly labeled and tetramerpositive cells were enriched from lymph nodes and spleen or lung aspreviously described (Hondowicz, Immunity, 2016). Both the columnflow-through and bound fractions were saved for flow cytometry analysis.Cells were surface stained with antibodies and analyzed on a BD LSR 1Iflow cytometer (BD Biosciences). Animal models: C57BL/6 mice werepurchased from The Jackson Laboratory or bred in house and. BALB/c micewere purchased from Charles River. Animals were maintained according toprotocols approved by Dana-Farber Cancer Institute (DFCI) InstitutionalAnimal Care and Use Committee, Direção Geral de Veterinária and iMMLisboa ethical committee.

Colorectal carcinoma in vivo mice experiments: CT26 cells were sourcedfrom Jocelyne Demengeot's research group at IGC (Instituto Gulbenkian deCiencia), Portugal. On day 0, 5×10{circumflex over ( )}5 cells wereinjected subcutaneously (s.c.) into the flanks of BALB/c mice with 50 μLof a 1:1 mixture of Dulbecco's modified Eagle medium (Gibco) withMatrigel (Corning). Starting on day 6, when tumour volume reached around100 mm3, neoleukin-2/15 and mIL-2 (Peprotech) were administered daily byintraperitoneal (i.p.) injection in 50 μL of PBS (Gibco). Treatment withanti-PD-1 antibody (Bio X Cell) was performed twice a week by i.p.injection of 200 μg per mouse in PBS. Mice were sacrificed when tumourvolume reached 1,300 mm3.

Melanoma in vivo experiments: B16F10 cells were purchased from ATCC. Onday 0, 5×10⁵ cells were inoculated by s.c. injection in 500 μL of Hank'sBalanced Salt Solution (Gibco). Starting on day 1, neoleukin-2/15 andmiL-2 (Peprotech) were administered daily by intraperitoneal (i.p.)injection in 200 μL of LPS-free PBS (Teknova). Treatment with TA99 (agift from Noor Momin and Dane Wittrup, Massachusetts Institute ofTechnology) at 150 μg/mouse was added several days later as indicated.Mice were sacrificed when tumor volume reached 2,000 mm3.

Flow cytometry: Excised tumors were minced, enzymatically digested(Miltenyi Biotec), and passed through a 40-μm filter. Cells from spleensand tumor-draining lymph nodes were dispersed into PBS through a 40-μmcell strainer using the back of a 1-mL syringe plunger. All cellsuspensions were washed once with PBS, and the cell pellet wasresuspended in 2% inactivated fetal calf serum containingfluorophore-conjugated antibodies. Cells were incubated for 15 minutesat 4° C. then fixed, permeabilized, and stained using a BioLegend FoxP3staining kit. Samples were analyzed on a BD Fortessa flow cytometer.Antibodies (BioLegend) used in melanoma experiments were: CD45-BV711(clone 30-F11), CD8-BV650 (53-6.7), CD4-BV421 (GK1.5), TCRβ-BV510(H57-597), CD25-AF488 (PC61), FoxP3-PFE (MF-14). Antibodies(eBioscience) used in colon carcinoma experiments were: CD45-BV510(30-F11), CD3-BV711 (17A2), CD49b-FTTC (DX5), CD4-BV605 (GK1.5),CD8-PECy7 (53-6.7), Foxp3-APC (FJK-16s). Fixable Viability Dye eFluor780 (eBioscience) was used to exclude dead cells.

Generation of anti-neoleukin-2/15 polyclonal antibody: Mice wereinjected i.p, with 500 μg of K.O. neoleukin in 200 μL of a 1:1 emulsionof PBS and Complete Freund's Adjuvant. Mice were boosted on days 7 and15 with 500 μg of K.O. neoleukin in 200 μL of a 1:1 emulsion of PBS andIncomplete Freund's Adjuvant. On day 20, serum was collected andrecognition of neoleukin-2/15 was confirmed by ELISA.

Enzyme-linked immunosorbent assay (ELISA): High-binding 96-well plates(Corning) were coated overnight at 4° C. with 100 ng/miL ofneoleukin-2/15, mIL-2 (Peprotech), hIL-2 (Peprotech), or ovalbumin(Sigma-Aldrich) in carbonate buffer. Antibody binding to target proteinswas detected using HRP-conjugated sheep anti-mouse IgG (GE Healthcare)at 75 ng/mL. Plates were developed with tetramethylbenzidine and HCLAbsorbance was measured at 450 nm with an EnVision Multimode PlateReader (PerkinElmer).

T cell proliferation assay: Cells were isolated from a mouse spleenusing an EasySep T Cell Isolation Kit (Stemcell Technologies). They wereplated in RPMI in 96-well culture plates at a density of 10,000cells/well. Media were supplemented with regular or heat-treatedneoleukin-2/15, rmIL-2, or Super-2. After 5 days of incubation at 37° C.cell survival and proliferation were measured by CellTiter-GloLuminescent Cell Viability Assay (Promega).

Statistical and power analyses: In vivo murine airway inflammationexperiments: MIKEL. In vivo murine Colon cancer experiments: CARLOS. Invivo murine Melanoma experiments: Comparisons of the survival oftumor-bearing mice were performed using the log-rank (Mantel-Cox) test.Comparisons of weight loss in tumor-bearing mice were performed using atwo-tailed t test. A P value less than 0.05 was considered to besignificant. The minimum group size was determined using G*Power for anexpected large effect size (Cohen's d=75).

Biolayer Interferometry analysis of a Mouse Serum Albumin (MSA) fusionto Neoleukin-2/15. Genetic fusion of Neoleukin-2/15 to MSA for extendedhalf-life and preserves intact binding affinity of the cytokine mimeticto murine IL-2RBeta and IL-2RGamma (33.5±0.2 nM) (data not shown). Theconstruct utilized in this study was as follows:

Optional: (HisTag TEV cleavage site in parentheses)Mouse serum albumin (italicized) Linker Neo2/15 (bold font)(SEQ ID NO: 244) (GSDGGSHHHHHHGSGSENLYFQGSG)EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNIRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKINCDLYEKLGEYGFQNAILVRYTOKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICILPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGP NLVTRCKDALAGGGSGGSGGGSGGSGSG PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS

Biotin-mIL2Gamma was immobilized on a Streptavidin biosensor, MSA-Neo2concentration was titrated from 729 to 1 nM in presence of saturatingconcentrations of mIL2Beta. Biolayer interferometry was carried out asabove: binding data were collected in a Octet RED96 (ForteBio, MenloPark, Calif.) and processed using the instrument's integrated softwareusing a 1:1 binding model. Biotinylated target receptors, either humanor murine IL-2Rα, IL-2β, IL-2

, or human IL-4Rα, were functionalized to streptavidin coated biosensors(SA ForteBio) at 1 μg/ml in binding buffer (10 mM HEPES [pH 7.4], 150 mMNaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk) for 300seconds. Analyte proteins were diluted from concentrated stocks intobinding buffer. After baseline measurement in binding buffer alone, thebinding kinetics were monitored by dipping the biosensors in wellscontaining 100 nM of the designed protein (association) and then dippingthe sensors back into baseline wells (dissociation).

CAR-T cell in vivo experiments: In vitro T cell proliferation assay.Primary human T cells were obtained from healthy donors. Peripheralblood mononuclear cells (PBMC) were isolated by centrifugation overFicoll-Hypaque (Sigma). T cells were isolated using EasySep™ CD8 or CD4negative isolation kits (STEMCELL Technologies). To stimulate T cells, Tcells were thawed and incubated with anti-CD3/CD28 Dynabeads (Gibco) at1:1 ratio in media supplemented with 50 IU/ml (3.1 ng/ml) of IL2. Beadswere removed after four days of incubation. Stimulated or freshly thawedunstimulated T cells were plated at 30000 or 50000 cells/well,respectively, in 96 well format and cultured in indicated concentrationsof IL2 or neoleukin-2/15 in triplicate. Three days later, proliferationwas measured using CellTiter-Glo 2.0. (Promega).

In vivo RAJI experiment: Six- to eight-week old NSG mice were obtainedfrom the Jackson Laboratory. 0.5*10{circumflex over ( )}6 RAJI tumorcells transduced with ffluc/eGFP were tail vein injected into the NSGmice. Seven days post tumor inject, lentiviral transduced anti-CD19 CART cells (0.4*10{circumflex over ( )}6 CD4, 0.4*10{circumflex over ( )}6CD8) prepared as described in (Liu et al, 2016) were infused i.v. intomice. hIL2 or neoleukin-2/15 at 20 μg/mouse were given i.p. from day 8to 16 post tumor injection.

Preparation of PEGylated polypeptides: Neo-2/15 stocks with eithersingle or dual cysteine mutations were dialyzed into phosphate buffer,pH7.0 and adjusted to 1.0-2.0 mg/ml. TCEP was added at a molar ratio of10:1 to protein and incubated for 10 minutes at RT to reduce disulfides.Maleimide-modified PEG40k (PEG40k-MA) or PEG30k (PEG30k-MA) powder wasadded directly to the reduced protein solution at a molar ratio of 10:1PEG:cysteine and incubated for 2 hours with stirring. Aliquots forSDS-PAGE were taken directly from the reaction mixture. These datademonstrate the rapid, spontaneous, and near-quantitative formation ofcovalent linkages between PEG40k-MA or PEG30k-MA and Neo-2/15 cysteinemutants in the expected stoichiometry.

Treatment with Neo-2/15 and PEGylated Neo-2/15-E62C (Neo-2/15-PEG)demonstrated changes in the levels of multiple inflammatory markers: Twonon-human primates (NHP), one male and one female per group, wereassigned to treatment with either vehicle (group 1), Neo-2/15 (w/o PEG)(groups 2-4) or Neo-2/15 PEG (groups 5-7; single cysteine mutation ofE62C and PEG40K). Animals treated with vehicle or Neo-2/15 (w/o PEG)were dosed by intravenous (IV) bolus on study days 1, 2, 3, 4, 5, 6 and7 (once daily for one week) at dose levels of either 0 (vehicle) or doseadjusted values of 0.07, 0.21 or 0.14 mg/kg/day Neo 2/15 (w/o PEG)(groups 2, 3 and 4, respectively). Animals treated with Neo-2/15 PEGwere dosed by IV bolus on study days 1 and 7 at dose levels of 0.05,0.15 or 0.10 mg/kg/day Neo-2/15PEG (groups 5, 6 and 7, respectively).Cytokine samples were taken on day 1 and 7 at timepoints of 0, 4, 8 and24 hours post dose. Cytokine serum samples were prepared and frozen at<−70° C. and shipped for analysis where samples were analyzed through aLuminex multiplex immunoassays system. Several cytokines, includingIL-15 and IL-10 demonstrated marked differences in the time-course ofcytokine production, consistent with a more sustained pharmacodynamiceffect for the PEGylated molecule.

Targeted Neo-2/15 fusions retained their IL-2R binding affinity anddemonstrated anti-tumor effects. Select targeting domains were fused tothe N- or C-termini of Neo-2/15 via peptide linkers and were tested invitro to characterize their binding affinity to human and mouse IL-2R byBiolayer Interferometry. The results confirmed that fusions to Neo-2/15at either the N or C termini did not hinder its ability to bind IL-2R.Subsequent in vitro Flow Cytometry studies confirmed that the fusionproteins were capable of binding a target receptor on the surface of acell. The efficacy of the targeted constructs was evaluated in in vivomouse experiments, in which it was demonstrated that a targeted Neo-2/15moiety to tumor cells or immune cells has a beneficial anti-tumor effectover a non-targeted control (data not shown).

Fusions that were tested include but are not limited to: (i) a fusion ofan anti-CD47 nanobody to the C terminus of Neo 2/15 via the linker ofSEQ ID NO:100; (b) a fusion of an anti-CD47 nanobody to the N terminusof Neo 2/15 via the linker of SEQ ID NO:100; (c) a fusion of ananti-CTLA4 nanobody to the C terminus of Neo 2/15 via the linker of SEQID NO:100; (d) a fusion of anti-CTLA4 nanobody to the N terminus of Neo2/15 via the linker of SEQ ID NO: 100; (e) a fusion of an anti-PDL-1nanobody to the C terminus of Neo 2/15 via the linker of SEQ ID NO:100;and (f) a fusion of an anti-PDL-1 nanobody to the N terminus of Neo 2/15via the linker of SEQ ID NO:100.

Fusions of albumin to Neo-2/15 maintained IL-2R binding affinity. Mouseserum albumin (MSA) was fused to the N-terminus of Neo 2/15 via apeptide linker and was tested in vitro to characterize its bindingaffinity to mouse IL-2R by Biolayer Interferometry. Biotin-mIL2Gamma wasimmobilized on a Streptavidin biosensor, MSA-Neo2 concentration wastitrated from 729 to 1 nM in presence of saturating concentrations ofmIL2Beta. The fusions maintained IL-2R binding capacity (data notshown).

PEGylated and non-PEGylated Neo-2/15 does not elicit a meaningfulanti-drug antibody (ADA) response in non-human primates (NHPs). Thepotential of PEGylated and non-PEGylated Neo-2/15 (for PEGylatedNeo-2/15: single cysteine mutation of E62C and PEG40K) to elicit ADAswas tested in non-human primates. Animals were administeredintravenously with either compound for 1 week: PEGylated Neo-2/15 ondays 1 and 7; wild-type Neo-2/15 on days 1-7. Blood was drawn at varioustimes thereafter and analyzed for the presence of antibodies specificfor the administered compound. Each dose group consisted of 1 male and 1female macaque. Non-PEGylated Neo-2/15 was administered via daily ivbolus injection for 7 consecutive days at 0.1m/kg, 0.2 mg/kg, or 0.3mg/kg. PEGylated Neo-2/15 was administered via iv bolus injection at0.015 mg/kg, 0.050 mg/kg, and 0.10 mg/kg on days 1 and 7. An equivalentvolume of saline was administered daily to a vehicle control group for 7consecutive days. Approximately 750 ul of blood was collected from eachanimal for ADA analysis on study Days 1 (pre-dose), 22, 29, and 43 viathe cephalic or saphenous vein. Serum was extracted from blood using aserum separator tube on wet ice and subsequently stored at −80 C untilanalysis. All cynomolgus macaques receiving either vehicle or PEGylatedNeo-2/15 tested negative for ADAs on days 22, 29, and 43 demonstratingthat PEGylated Neo-2/15 did not elicit a detectable immune response,even after repeat dosing, despite being a computationally-designedprotein that is entirely foreign to the macaque immune system. Both (1male; 1 female) macaques receiving vehicle control tested negative forADAs against wild-type Neo-2/15 on days 1, 15, 22, and 28. All animals(3 males; 2 females) in the groups receiving non-PEGylated Neo-2/15tested negative for ADAs on day 1 (pre-dose). Of these, 3 out of 5 (60%)remained negative for ADAs on days 22, 29, and 43. The remaining twoanimals subsequently tested positive for ADAs on days 22, 29, or 43. Onesubject tested positive on days 22 and 29, but returned negative by day43. For that subject, the ADA response was low and transient, suggestingminimal clinical significance. Another subject tested positive on days22, 29, and 43. For that subject, the measured ADA concentrations werewell below 100 ng/ml and thus of unclear clinical relevance.

Data Tables

Table E1. Characterization of several de novo designed mimetics ofIL-2/IL-15. The table shows the Kd of de novo IL-2/IL-15 mimetics andreference cytokines for: mIL-2RO, mIL-2Rβ

c, EC₅₀, the sequence similarity by structural alignment (MICAN ⁶³)against hIL-2 (PDB: 2B51) and mIL-2 (PDB:), the parent of each molecule,its amino acid length, and the sequences for the de novo IL-2 mimetics.“N/S” stands for non-significant and “N/A” for non-available.

TABLE E1 Binding affinity (Kd) to HsIL-2Rβ 

 c, and cell signaling in human NK (YT, CD25−) cells EC50 Kd Kd (CD25−)Seq identity Seq identity HsIL- HsIL- pSTAT5p to HsIL-2 to MmIL-2 Exp.2Rβ 

 c 2Rβ (nM)/ (%/(num (%/(num opti- a.a. Name (nM) (nM) (exp i.d.) a.a.aIgn)) a.a. aIgn)) mized Parent molecule length HsIL-2 193.6 326.90.41/(a) 100.0/(120)  54.5/(112) — — 133 MmIL-2 8034.0 4950.0 39.05/(a)  54.5/(112)   100/(122) — — 130 Super-2/ 300.9 2.0 0.07/(a)  94.9/(117) 50.9/(114) Y HsIL-2 133 Superkine (PDB: 3QAZ) G1_neo2_40 260.0 1457.00.14/(b) 47.7/(86) 30.4/(79) N — 87 G1_neo2_41 187.0 720.6 0.07/(b)47.7/(86) 30.4/(79) N — 87 G1_neo2_43 533.4 2861.0 0.21/(b) 50.0/(86)32.9/(79) N — 87 G1_neo2_40_1F 2.3 2.6 0.09/(c) 44.2/(86) 26.6/(79) YG1_neo2_40 87 G2_neo2_40_1F_dsn36 113.9 27.6 0.12/(a) 33.7/(89)17.6/(85) N De novo mimetic 100 design inspired on template:G1_neo2_40_1F Neoleukin-2/15 18.8 11.2 0.05/(a) 29.2/(89) 15.7/(83) YG2_neo2_40_1F_dsn36 100 (G2_neo2_40_1F_dsn36_opt) Binding affinity (Kd)to MmIL-2Rβ 

 c, and cell signaling (EC50) in murine T (CTLL-2, CD25+) cells EC50 KdKd (CD25+) Seq identity Seq identity MmIL- MmIL- pSTAT5 to HsIL-2 toMmIL-2 Exp. 2Rβ 

 c 2Rβ (nM)/ (%/(num (%/(num opti- a.a. Name (nM) (nM) (exp i.d.) a.a.aIgn)) a.a. aIgn)) mized Parent molecule length HsIL-2 492.2 8106.00.002/(d)  *see top table MmIL-2 126.2 1496.0 0.003/(e)  *see top tableSuper-2/ 312.2 214.0  N/A *see top table Superkine (PDB: 3QAZ)G1_neo2_40_1F 7.9 485.5   0.2/(e) *see top table G1_neo2_40_1F_H1 2654.06799.0 37.38/(d)  39.5/(86) 25.0/(80) Y G1_neo2_40_1F 87G1_neo2_40_1F_H2 963.7 68300.0  9.38/(d) 40.7/(86) 26.2/(80) YG1_neo2_40_1F 87 G1_neo2_40_1F_H3 3828.0 N/S  35.2/(d) 39.5/(86)25.0/(80) Y G1_neo2_40_1F 87 G1_neo2_40_1F_H4 391.8 10070.0  0.93/(d)41.9/(86) 26.2/(80) Y G1_neo2_40_1F 87 G1_neo2_40_1F_H5 5123.0 45300.084.69/(d)  39.5/(86) 23.8/(80) Y G1_neo2_40_1F 87 G1_neo2_40_1F_M1 4.3213.9 0.007/(d)  36.0/(86) 25.0/(80) Y G1_neo2_40_1F 87 G1_neo2_40_1F_M2886.3 2599.0  3.11/(d) 37.2/(86) 25.0/(80) Y G1_neo2_40_1F 87G1_neo2_40_1F_M3 64.8 402.3  0.08/(d) 34.9/(86) 25.3/(79) YG1_neo2_40_1F 87 G2_neo2_40_1F_seq04 80.0 N/A 1.95/(f) 38.4/(86)23.8/(80) N Sequence 87 redesign of G1_neo2_40_1F G2_neo2_40_1F_seq1239.1 N/A 1.74/(f) 38.4/(86) 25.3/(79) N Sequence 87 redesign ofG1_neo2_40_1F G2_neo2_40_1F_seq16 71.5 N/A 2.20/(f) 34.9/(86) 22.5/(80)N Sequence 87 redesign of G1_neo2_40_1F G2_neo2_40_1F_seq26 27.8 N/A1.06/(f) 39.5/(86) 25.3/(79) N Sequence 87 redesign of G1_neo2_40_1FG2_neo2_40_1F_seq27 13.6 N/A 0.24/(f) 36.0/(86) 25.0/(80) N Sequence 87redesign of G1_neo2_40_1F G2_neo2_40_1F_dsn29 38.2 N/A 0.48/(f)36.6/(82)  8.9/(90) N De novo mimetic 107 design using template:G1_neo2_40_1F G2_neo2_40_1F_dsn30 925.0 N/A 7.61/(f) 33.0/(97) 23.4/(94)N De novo mimetic 107 design using template: G1_neo2_40_1FG2_neo2_40_1F_dsn36 568.5 2432.0  1.36/(e) *see top tableG2_neo2_40_1F_dsn40 69.2 N/A 0.50/(f) 33.7/(89) 17.9/(84) N De novomimetic 100 design inspired on template: G1_neo2_40_1F Neoleukin-2/1538.4 16.1  0.07/(e) *see top table (G2_neo2_40_1F_dsn36_opt)

TABLE E2 Crystallographic data table for neoleukin-2/15 andneoleukin-2/15 quaternary complex with mIL-2Rβγ_(c). Neoleukin-2/15ternary Neoleukin-2/15 complex with IL-2R (6DG6) (6DG5) WavelengthResolution range 39.28-1.999 (2.07-1.999) 47.005-2.516 (2.828-2.516)Ellipsoidal resolution limit (Å) — 3.687 (0.065 a* + 0.998 c*)(direction) — 3.756 (0.884 a* + 0.468 c*) — 2.516 (0.132 a* + 0.859 b* +0.495 c*) Space group P 21 21 21 P 21 2 21 Unit cell (Å, °) 73.73, 86.8,92.31, 90, 90, 90 65.125, 67.914, 172.084, 90, 90, 90 Total reflections351741 (32344) 132356 (7834) Unique reflections 40650 (3977) 13961 (698)Multiplicity 8.7 (8.1) 9.5 (11.2) Completeness (spherical) (%) 92.58(77.83) 52.3 (9.0) Completeness (ellipsoidal) (%) 93.2 (77.2) MeanI/sigma(I) 12.19 (1.25) 6.8 (1.3) Wilson B-factor 34.54 39.86 R-merge0.1027 (1.709) 0.359 (2.516) R-meas 0.1094 (1.824) 0.380 (2.636) R-pim0.0369 (0.6252) 0.122 (0.780) CC1/2 0.999 (0.557) 0.987 (0.445) CC* 1(0.846) 0.993 (0.328) Resolution range used in refinement 39.28-1.999(2.07-1.999) 43.82-2.516 (2.606-2.516) Reflections used in refinement37747 (3125) 13923 (136) Reflections used for R-free 1840 (143) 1366(14) R-Work 0.2037 (0.3137) 0.2211 (0.3271) R-free 0.2260 (0.3377)0.2658 (0.4429) Number of non-hydrogen atoms 4791 4100 macromolecules4735 3949 ligands — 138 solvent 56 13 Protein residues 597 492RMS(bonds) 0.005 0.004 RMS(angles) 0.88 0.94 Ramachandran favored (%)97.41 97.1 Ramachandran allowed (%) 2.59 2.9 Ramachandran outliers (%) 00 Rotamer outliers (%) 1.26 4.5 Clashscore 2.14 4.55 Average B-factor52.56 47.05 macromolecules 52.54 46.39 ligands — 67.79 solvent 54.2127.31 Number of TLS groups 20 3 *Statistics for the highest-resolutionshell are shown in parentheses.

TABLE S1 Amino acid sequences for the best twelve first-round designs.Ten of the designs were (G1_neo2_35-44) were experimentallycharacterized by yeast display and all but two (G1_neo2_35and G1_neo2_44) were found to bind fluorescently labeledchimeric ILRβγc at low nanomolar concentrations via flowcytometry screening of designed first-round protein binders.Designs indicated were expressed on yeast and incubated with 2nM hIL-2Rβ

_(c) or 0 nM IL-2Rβ

_(c) (data not shown). Design Sequence G1_neo2_33STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDLDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 103) G1_neo2_34STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSCISTGKCDLDKAEDIRNSDQARREAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 104) G1_neo2_35STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDCDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAC (SEQ ID NO: 105) G1_neo2_36STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 106) G1_neo2_37STKKLQLQAEHFLLDVQMILNESPEPNEELNRCITDAQSWISTGKIDLDRAEECARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 107) G1_neo2_38STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSCISTGKCDLDRAEELARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 108) G1_neo2_39STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELCRNLEKVRDEALKRGIDVRDLVSNACVIALELK (SEQ ID NO: 109) G1_neo2_40STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSWISTGKIDLDGAKELAKEVEELRQEAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 110) G1_neo2_41STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSCISTGKCDLDGAKELAKEVEELRQEAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 111) G1_neo2_42STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMAKEAEKIRKEMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 112) G1_neo2_43STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSCISTGKCDLDNAQEMAKEAEKIRKEMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 113) G1_neo2_44STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMCKEAEKIRKEMEKRGIDVRDLISNICVILLELS (SEQ ID NO: 114)

TABLE S2Amino acid sequences for the experimentally optimized first-rounddesigns. Design Sequence G1_neo2_STKKTQLLAEHALLDAFMMLNVVPEPNEKLNRIITTMQSWITTGKIDADGAKELAKEVEELE 40_1AQEYEKRGIDVEDDASNLKVILLELA (SEQ ID NO: 115) G1_neo2_STKKTQLLAEHALLDAHMMLNMLPEPNEKLNRIITTMQSWIHTGKIDGDGAQELAKEVEELE 40_1BQEYEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 116) G1_neo2_STKKTQLLAEHALLDAFMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELE 40_1CQEFEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 117) G1_neo2_STKKTQLLAEHALLDALMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELE 40_1D_QELEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 118) G1_neo2STKKTQLLAEHALLDAHMMLNVVPEPNEKLNRIITTMQSWITTGKIDRDGAQELAKEVEELE 40_1EQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 119) G1_neo2_STKKTQLLAEHALLDALMMLNLLPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELE 40_1FQEHEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 120) G1_neo2_STKKTQLLAEHALLDAYMMLNMVPEPNEKLNRIITTMQSWILIGKIDSDGAQELAKEVEELE 40_1GQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 121) G1_neo2_STKKTHLLAEHALLDAYMMLNVMPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELE 40_1HQEFEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 122) G1_neo2_STKKTQLLAEHALLDAYMMLNLVPEPNEKLNRIITTMQSWIFTGKIDADGAQELAIEVEELE 40_1IQEYEKRGIDVDDYASNLKVILLELA (SEQ ID NO: 123) G1_neo2_STKKTQLMAEHALLDAFMMLNVLPEPNEKLNRIITTMQSWIFTGKIDGDDAQELAKEVEELE 40_1JQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 124) G1_neo2_STKKTQLLIEHALLDALDMSRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQQLAKEVEELE 40_1F_H1QEHEKRGEDVEDEASNLKVILLELA (SEQ ID NO: 125) G1_neo2_STKKTQLLLEHALLDALHMRRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQELAKEVEELE 40_1F_H2QEHEKRGRDVEDDASNLKVILLELA (SEQ ID NO: 126) G1_neo2_STKKTQLLIEHALLDALNMRKKLPEPNEKLSRIITDMQSWIFTGKIDGDGAQQLAKEVEELE 40_1F_H3QEHEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 127) G1_neo2_STKKTQLLLEHALLDALHMSRELPEPNEKLNRIITDMQSWIFTGKIDGDGAQDLAKEVEELE 40_1F_H4QEHEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 128) G1_neo2_STKKTQLLIEHALLDALHMSRKLPEPNEKLSRIITTMQSWIFTGKIDGDGAQHLAKEVEELE 40_1F_H5QEHEKRGGEVEDEASNLKVILLELA (SEQ ID NO: 129) G1_neo2_STKKTQLLIEHALLDALHMKRKLPEPNEKLNRIITNMQSWIFTEKIDGDGAQDLAKEVEELE 40_1F_H6QEHEKRGQDVEDYASNLKVILLELA (SEQ ID NO: 130) G1_neo2_STEKTQLAAEHALRDALMLKHLLNEPNEKLARIITTMQSWQFTGKIDGDGAQELAKEVEELQ 40_1F_M1QEHEVRGIDVEDYASNLKVILLHLA (SEQ ID NO: 131) G1_neo2_STKNTQLAAEDALLDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQ 40_1F_M2QEHEERGIDVEDYASNLKVILLQLA (SEQ ID NO: 132) G1_neo2_STEKTQHAAEDALRDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQ 40_1F_M3QEHEVRGIDVEDYASNLKVILLQLA (SEQ ID NO: 133)

TABLE S3 Amino acid sequences for second-round designs.G2neo2_40_1F_seq02 to G2_neo2_40_1F seq28 correspond to the 27Rosetta sequence redesigns of G1_neo2_40_1F; G2_neo2_40_1F_seq29to G2_neo2_40_1F_seq42 represent the 14 new de novo mimetic designs.Design Sequence G2_neo2_40_TQKKQQLLAEHALLDALMILNMLKTSSEAVNRMITIAQSWIFTGTSNPEEAKEMIKMA 1F_seq02EQAEEEARREGVDTEDYVSNLKVILKEIA (SEQ ID NO: 134) G2_neo2_40_TTKKYQLLVEHALLDALMMLNLSSESNEKMNRIITTMQSWIFTGTFDPDQAEELAKLV 1F_seq03EELREEFRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 135) G2_neo2_40_TTKKIQLLVEHALLDALMILNLSSESNEKLNRIITTLQSWIFRGEIDPDPARELAKLL 1F_seq04EEIREEMRKRGIDTEDYVSNMIVIIRELA (SEQ ID NO: 136) G2_neo2_40_TKKKIQLLAEHVLLDLLMMLNLSSESNEKMNRLITIVQSWIFTGTIDPDQAEEMAKWV 1F_seq05EELREEFRKRGIDTEDYASNVKVILKELS (SEQ ID NO: 137) G2_neo2_40_TKKKYQLLIEHLLLDALMVLNMSSESNEKLNRIITILQSWIFTGTWDPDLAEEMEKLM 1F_seq06QEIEEELRRRGIDTEDYMSNMRVIIKELS (SEQ ID NO: 138) G2_neo2_40_TKKKLQLLVEHLLLDMLMILNMSSESNEKLNRLITELQSWIFRGEIDPDKAEEMWKIM 1F_seq07EEIEKELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 139) G2_neo2_40_TSKKQQLLAEHALLDALMILNISSESSEAVNRAITWLQSWIFKGTVNPDQAEEMRKLA 1F_seq08EQIREEMRKRGIDTEDYVSNLEVIAKELS (SEQ ID NO: 140) G2_neo2_40_TKKKYQLLIEHLLLDLLMVLNMSSESNEKINRLITWLQSWIFTGTYDPDLAEEMYKIL 1F_seq09EELREEMRERGIDTEDYMSNMRVIVKELS (SEQ ID NO: 141) G2_neo2_40_TKKKWQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFTGTYDPDLAEEMKKMM 1F_seq10DEIEDELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 142) G2_neo2_40_TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELSKLV 1F_seq11EEIREEMRKRGIDTEDYVSNLKVILDELS (SEQ ID NO: 143) G2_neo2_40_TEKKLQLLVEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGRIDPDKAEELAKLV  1F_seq12EELREEARERGIDTEDYVSNLKVILKELS (SEQ ID NO: 144) G2_neo2_40_TKKKYQLLMEHLLLDLLMVLNMSSESNEKLNRLITIIQSWIFTGTWDPDKAEEMAKML 1F_seq13KEIEDELRERGIDTEDYMSNMIVIMKELS (SEQ ID NO: 145) G2_neo2_40_TTKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFEGRIDPDQAQELAKLV  1F_seq14EELREEFRKRGIDTEDYVSNLKVILEELS (SEQ ID NO: 146) G2_neo2_40_TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELAKLV 1F_seq15RELREEFRKRGIDTEDYASNLEVILRELS (SEQ ID NO: 147) G2_neo2_40_TKKKIQLLVEHALLDALMILNLSSKSNEKLNRIITTMQSWIFNGTIDPDRARELAKLV 1F_seq16EEIRDEMEKNGIDTEDYVSNLKVILEELA (SEQ ID NO: 148) G2_neo2_40_TKKKYQLLIEHVLLDLLMLLNLSSESNEKMNRLITILQSWIFTGTYDPDKAEEMAKLL 1F_seq17KELREEFRERGIDTEDYISNAIVILKELS (SEQ ID NO: 149) G2_neo2_40_TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLV 1F_seq18EELREEFRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 150) G2_neo2_40_TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFNGTIDPDQARELAKLV 1F_seq19EELREEFRKRGIDTEDYASNLKVILEELA (SEQ ID NO: 151) G2_neo2_40_TKKKLQLLVEHALLDALMLLNLSSESNEKLNRIITTMQSWIFTGTVDPDQAEELAKLV 1F_seq20EEIREELRKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 152) G2_neo2_40_TTKKYQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTFDPDQAEELAKLV 1F_seq21REIREEMRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 153) G2_neo2_40_TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLV 1F_seq22REIREEMRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 154) G2_neo2_40_TKKKYQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFRGEWDPDKAEEWAKIL 1F_seq23KEIREELRERGIDTEDYMSNAIVIMKELS (SEQ ID NO: 155) G2_neo2_40_TDKKLQLLVEHLLLDLLMMLNLSSKSNEKMNRLITIAQSWIFTGKVDPDLAREMIKLL 1F seq24EETEDENRKNGIDTEDYVSNARVIAKELE (SEQ ID NO: 156) G2_neo2_40_TKKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFTGTIDPDQAEELAKLV 1F_seq25EELKEEFKKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 157) G2_neo2_40_TKKKYQLLIEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGTYDPDKAEELEKLA 1F_seq26KEIEDEARERGIDTEDYMSNLRVILKELS (SEQ ID NO: 158) G2_neo2_40_TKKKAQLLAEHALLDALMLLNLSSESNERLNRIITWLQSIIFTGTYDPDMVKEAVKLA 1F_seq27DEIEDEMRKRGIDTEDYVSNLRVILQELA (SEQ ID NO: 159) G2_neo2_40_TQKKNQLLAEHLLLDALMVLNQSSESSEVANRIITWAQSWIFEGRVDPNKAEEAKKLA 1F_seq28KKLEEEMRKRGIDMEDYISNMKVIAEEMS (SEQ ID NO: 160) G2 neo2_40_EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLN 1F_seq29RLITYIQSQIFEISERIRETDQEKKEESWKKWQLLLEHALLDVLMLLND (SEQ ID NO: 161)G2_neo2_40_ PEKKRQLLLEHILLDALMLLNLLETNPQNTESKFEDYISNAEVIAEELAKLMESLGLS1F_seq30DEAEKFKKIKQWLREVWRIWSSTNWSTLEDKARELLNRIITTIQSQIFY (SEQ ID NO: 162)G2_neo2_40_ PEKKRQLLLEHILLDLLMILNMIETNRENTESEMEDYWSNVRVILRSLAKLMEELNYK1F_seq31 ELSELMERMRKIVEKIRQIVTNNSSLDTAREWLNRLITWIQSLIFR (SEQ ID NO: 163)G2_neo2_40_ PEKKRQLLAEHALLDALMLLNIIETNSKNTESKMEDYVSNLEVILTEFKKLAEKLNPS1F_seq32 EEAERAERMKPWARKAYQMMTLDLSLDKAKEMLNRIITILQSIIFN (SEQ ID NO: 164)G2_neo2_40_ PEKKRQLLAEHLLLDVLMMLNGNASLKDYASNAQVIADEFRELARELGLTDEAKKAEK1F_seq33 IIEALERAREWLLNNKDKEKAKEALNRAITIAQSWIFN (SEQ ID NO: 165)G2_neo2_40_ PEKKRQLLLEHLLLDLLMILNMLRTNPKNIESDWEDYMSNIEVIIEELRKIMESLGRS1F_seq34 EKAKEWKRMKQWVRRILEIVKNNSDLEEAKEWLNRLITIVQSFIFE (SEQ ID NO: 166)G2_neo2_40_ WEKKRQLLLSHLLLDLLMILNMWRTNPQNTESLMEDYMSNAKVIVESLARMMRSQGLE1F_seq35 DKAREWEEMKKRIEEIRQIIQNNSSKERAKEELNRLITYVQSEIFR (SEQ ID NO: 167)G2_neo2_40_ PKKKIQLLAEHALLDALMILNIVKTNSQNAEEKLEDYASNVEVILEEIARLHESGDQK1F_seq36 DEAEKAKRMKEWMKRIKTTASEDEQEEMANRIITLLQSWIFS (SEQ ID NO: 168)G2_neo2_40_ PEKKRQLLAEHALLDALMILMILQTNPQNAEEKLEDYMSNVEVIMEEFARMMRNGDRS1F_seq37 EEAENAERIKKWVRKASSTASSEEQREMMNRAITLMQSWIFE (SEQ ID NO: 169)G2_neo2_40_ PEKKRQLLAEHLLLDALMVLNMLTTNSKNTEEKLEDYISNMKVIIKEMIELMRSLGRL1F_seq38 EEAEKWKEALKAVSKIGSRMDSETARELANRIITLAQSAIFY (SEQ ID NO: 170)G2_neo2_40_ PEKKRQLLAEHALLDALMFLNLVETNPDQAEEKIEDYASNLRVIAEELARLFENLGRL1F_seq39 DEAQKAKDIKELAERARSRVSSEKRKEAMNRAITILQSMIFR (SEQ ID NO: 171)G2_neo2_40_ PEKKRQLLAEHALLDALMILNIIRTNSDNTESKLEDYISNLKVILEEIARLMESLGLS1F_seq40 DEAEKAKEAMRLADKAGSTASEEEKKEAMNRVITWAQSWIFN (SEQ ID NO: 172)G2_neo2_40_ PEKKRQLLAEHALLDALMMLNILRTNPDNAEEKLEDYWSNLIVILREIAKLMESLGLT1F_seq41 DEAEKAKEAARWAEEARTTASKDQRRELANRIITLLQSWIFS (SEQ ID NO: 173)G2_neo2_40_ PEKKRQLLAEHLLLDALMILNIIETNEQNAESKLEDYISNAKVILDEFREMARDLGLL1F_seq42 DEAKKAEKMKRWLEKMRSNASSDERREWANRMITTAQSWIFN (SEQ ID NO: 174)

TABLE S4 Amino acid sequences for the experimentallyoptimized second-round designs. Design Sequence G2_neo2_40_1F_INKEAQLHAEFALYDALMLLNLSSESNERLNRIITWLQSIIFYETYDPDMVKEAV seq27_S18KLADEIEDEMRKRKIDTEDYVVNLRLILQELA (SEQ ID NO: 175) G2_neo2_40_1F_TKKDAELLAEFALYDALMLLNLSSESNERLNEIITWLQSIIFYGTYDPDMVKEAV seq27_S22KLADEIEDEMRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 176) G2_neo2_40_1F_INKKAQLHAEFALYDALMLLNLSSESNERLNDIITWLQSIIFTGTYDPDMVKEAV seq27_S24KLADEIEDEMRKRKIDTEDYVVNLRYILQELA (SEQ ID NO: 177) G2_neo2_40_1F_EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKE seq29_S6LLNRLITYIQSQIFEVLHGVGETDQEKKEESWKKWDLLLEHALLDVLMLLND (SEQ ID NO: 178)G2_neo2_40_1F_ EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKEseq29_S7 LLNELITYIQSQIFEVIEREGETDQEKKEESWKKWELHLEHALLDVLMLLND(SEQ ID NO: 179) G2_neo2_40_1F_EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKE seq29_S8LLNRLITYIQSQIFEVLEGVGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 180)Neolukin-2/15 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESG(i.e. DQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO:G2_neo2_40_1F_ 181) seq36_S11) G2_neo2_40_1FPKKKIQLLAEHALFDLLMILNIVKTNSQNAEEKLEDYAYNAGVILEEIARLFESG seq36_S12DQKDEAEKAKRMKEWMKRIKDTASEDEQEEMANEIITILQSWNFS (SEQ ID NO:   182)Neoleukin-2/15-H8Y-K33E: H1->H3->H2′->H4PKKKIQLYAEHALYDALMILNIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS(SEQ ID NO: 94)

Binding of Neoleukin-2/15-H8Y-K33E to the IL2 receptor was measured bybiolayer interferometry, and it was found to have higher bindingaffinity than Neoleukin-2 for IL2-Rbeta, both when tested againstIL2Rbeta alone and when tested against the IL2Rbeta-gamma complex. Thisincreased affinity was attributable mostly to an improved off-rate fromIL2-Rbeta.

TABLE S5 Amino acid sequences for the interleukin-4mimetic designs based on reengineering of neolukin-2/15. Design SequenceIL4_G2_neo2_40_ PKKKIQITAEEALKDALSILNIVKINSPPAEE 1F_seq36_S11QLERFAKRFERNLWGIARLFESGDQKDEAEKA KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 183) Neoleukin-4 PKKKIQIMAEEALKDALSILNIVKINSPPAEE (i.e.QLERFAKRFERNLWGIARLFESGDQKDEAEKA IL4_G2_neo2_40_KRMIEWMKRIKTTASEDEQEEMANAIITILQS 1F_seq36_S11_MIF) WFFS (SEQ ID NO: 184)

1-138. (canceled)
 139. A method for treating cancer comprisingadministering to a subject having cancer a non-naturally occurringpolypeptide comprising domains X1, X2, X3, and X4, wherein: (a) X1 is apeptide comprising the amino acid sequence EHALYDAL (SEQ ID NO:1); (b)X2 is a helical-peptide of at least 8 amino acids in length; (c) X3 is apeptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:2); (d)X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ IDNO:3); wherein X1, X2, X3, and X4 may be in any order in thepolypeptide; wherein amino acid linkers may be present between any ofthe domains; and wherein the polypeptide binds to IL-2 receptor β

_(c) heterodimer (IL-2β

_(c)).
 140. The method of claim 139, wherein: X1 is a peptide comprisingan amino acid sequence at least 80% identical to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4) provided that the amino acids atpositions 10-17 relative to SEQ ID NO:4 are identical to SEQ ID NO:1; X3is a peptide comprising an amino acid sequence at least 80% identical tothe peptide LEDYAFNFELILEEIARLFESG (SEQ ID NO:5) provided that the aminoacids at positions 4-11 relative to SEQ ID NO:5 are identical to SEQ IDNO:2; and X4 is a peptide comprising an amino acid sequence at least 80%identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6) providedthat the amino acids at positions 12-20 relative to SEQ ID NO:6 areidentical to SEQ ID NO:3
 141. A method for treating cancer comprisingadministering to a subject having cancer a non-naturally occurringpolypeptide comprising domains X1, X2, X3, and X4, wherein: X1 is apeptide comprising an amino acid sequence at least 80% identical to thepeptide (SEQ ID NO: 4) PKKKIQLHAEHALYDALMILNI;

X2 is a helical-peptide of at least 8 amino acids in length; X3 is apeptide comprising an amino acid sequence at least 80% identical to thepeptide LEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptidecomprising an amino acid sequence at least 80% identical to the peptide(SEQ ID NO: 6) EDEQEEMANAIITILQSWIFS;

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;wherein amino acid linkers may be present between any of the domains;and wherein the polypeptide binds to IL-2 receptor β

_(c) heterodimer (IL-2Rβ

_(c)).
 142. The method of claim 141, wherein: X1 is a peptide comprisingthe amino acid sequence PKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is apeptide comprising the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQID NO:5); and X4 is a peptide comprising the amino acid sequenceEDEQEEMANAIITILQSWIFS (SEQ ID NO:6).
 143. The method of claim 141,wherein X2 is a peptide comprising an amino acid sequence at least 80%identical to the peptide KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7).
 144. Themethod of claim 142, wherein X2 is a peptide comprising an amino acidsequence at least 90% identical to the peptide KDEAEKAKRMKEWMKRIKT (SEQID NO:7).
 145. The method of claim 141 wherein the domains are arrangedN-terminal to C-terminal in an arrangement selected from X1-X3-X2-X4.146. The method of claim 144 wherein the domains are arranged N-terminalto C-terminal in an arrangement selected from X1-X3-X2-X4.
 147. Themethod of claim 139 wherein the polypeptide comprises an amino acidsequence at least 80% identical to the amino acid sequence set forth inSEQ ID NO:
 181. 148. The method of claim 147 wherein the polypeptidecomprises an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO:
 181. 149. The method of claim 148wherein the polypeptide comprises an amino acid sequence at least 95%identical to the amino acid sequence set forth in SEQ ID NO:
 181. 150.The method of claim 141 wherein the polypeptide comprises an amino acidsequence at least 80% identical to the amino acid sequence set forth inSEQ ID NO:
 181. 151. The method of claim 150 wherein the polypeptidecomprises an amino acid sequence at least 95% identical to the aminoacid sequence set forth in SEQ ID NO:
 181. 152. The method of claim 151wherein the polypeptide comprises the amino acid sequence set forth inSEQ ID NO:
 181. 153. The method of claim 151 wherein the polypeptidecomprises an amino acid sequence that is 99% identical to the amino acidsequence set forth in SEQ ID NO:181 and comprises a cysteine at position62.
 154. The method of claim 140 wherein the polypeptide is linked to astabilization compound.
 155. The method of claim 141 wherein thepolypeptide is linked to a stabilization compound.
 156. The method ofclaim 153 wherein a polyethylene glycol containing moiety is linked tothe cysteine residue at position
 62. 157. The method of claim 156wherein the polyethylene glycol is linked via a maleimide group to thecysteine residue.
 158. The method of claim 157 wherein the subject hasmelanoma or renal cell cancer.